Anti-CSF1R antibody and agonistic anti-CD40 antibody combination therapy for cancer

ABSTRACT

Methods of treating cancer with antibodies that bind colony stimulating factor 1 receptor (CSF1R) in combination with one or more immune stimulating agents are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/564,866, filed Oct. 6, 2017, which is a national stage application ofInternational Application No. PCT/US2016/027038, filed Apr. 12, 2016,which claims the benefit of priority of U.S. Provisional Application No.62/146,766, filed Apr. 13, 2015; and U.S. Provisional Application No.62/190,945, filed Jul. 10, 2015, each of which is incorporated byreference in its entirety for any purpose.

TECHNICAL FIELD

Methods of treating cancer with antibodies that bind colony stimulatingfactor 1 receptor (CSF1R) in combination with one or more immunestimulating agents.

BACKGROUND

Colony stimulating factor 1 receptor (referred to herein as CSF1R; alsoreferred to in the art as FMS, FIM2, C-FMS, M-CSF receptor, and CD115)is a single-pass transmembrane receptor with an N-terminal extracellulardomain (ECD) and a C-terminal intracellular domain with tyrosine kinaseactivity. Ligand binding of CSF1 or the interleukin 34 ligand (referredto herein as IL-34; Lin et al., Science 320: 807-11 (2008)) to CSF1Rleads to receptor dimerization, upregulation of CSF1R protein tyrosinekinase activity, phosphorylation of CSF1R tyrosine residues, anddownstream signaling events. CSF1R activation by CSF1 or IL-34 leads tothe trafficking, survival, proliferation, and differentiation ofmonocytes and macrophages, as well as other monocytic cell lineages suchas osteoclasts, dendritic cells, and microglia.

Many tumor cells or tumor stromal cells have been found to produce CSF1,which activates monocyte/macrophage cells through CSF1R. The level ofCSF1 in tumors has been shown to correlate with the level oftumor-associated macrophages (TAMs) in the tumor. Higher levels of TAMshave been found to correlate with poorer patient prognoses in themajority of cancers. In addition, CSF1 has been found to promote tumorgrowth and progression to metastasis in, for example, human breastcancer xenografts in mice. See, e.g., Paulus et al., Cancer Res. 66:4349-56 (2006). Further, CSF1R plays a role in osteolytic bonedestruction in bone metastasis. See, e.g., Ohno et al., Mol. CancerTher. 5: 2634-43 (2006). TAMs promote tumor growth, in part, bysuppressing anti-tumor T cell effector function through the release ofimmunosuppressive cytokines and the expression of T cell inhibitorysurface proteins. Thus, antibodies that bind to CSF1R may be useful inmethods of treating cancer.

Some tumor cells may escape detection by the immune system at least inpart by suppressing the immune response, for instance by altering theexpression of immune modulatory genes. For example, the relativeconcentrations of both immune stimulatory and immune inhibitorymolecules in the body may modulate the adaptive immune response. Arelatively high level of expression of inhibitory molecules and/orreduced expression of certain stimulatory molecules may create acheckpoint or switch that down-regulates the adaptive immune response.Agents that counteract this effect by up-regulating the adaptive immuneresponse or by stimulating the innate immune response are potentialcancer therapies.

A combination regimen of agents that modulate the immune response incancer, such as immune stimulating agents, may increase the depth anddurability of the response and may also broaden efficacy to patients whodo not respond to a single agent alone.

SUMMARY

In some embodiments, methods of treating cancer in a subject areprovided, comprising administering to the subject an anti-CSF1R antibodyand at least one immune stimulating agent. In some embodiments, the atleast one immune stimulating agent comprises an agonist of animmune-stimulatory molecule, including a co-stimulatory molecule, whilein some embodiments, the at least one immune stimulating agent comprisesan antagonist of an immune inhibitory molecule, including aco-inhibitory molecule. In some embodiments, the at least one immunestimulating agent comprises an agonist of an immune-stimulatorymolecule, including a co-stimulatory molecule, found on immune cells,such as T cells. In some embodiments, the at least one immunestimulating agent comprises an antagonist of an immune-inhibitorymolecule, including a co-inhibitory molecule, found on immune cells,such as T cells. In some embodiments, the at least one immunestimulating agent comprises an agonist of an immune-stimulatorymolecule, including a co-stimulatory molecule, found on cells involvedin innate immunity, such as NK cells. In some embodiments, the at leastone immune stimulating agent comprises an antagonist of animmune-inhibitory molecule, including a co-inhibitory molecule, found oncells involved in innate immunity, such as NK cells. In someembodiments, the combination enhances the antigen-specific T cellresponse in the treated subject and/or enhances the innate immunityresponse in the subject. In some embodiments, the combination results inan improved anti-tumor response in an animal cancer model, such as axenograft model, compared to administration of either the anti-CSF1Rantibody or immune stimulating agent alone. In some embodiments, thecombination results in a synergistic response in an animal cancer model,such as a xenograft model, compared to administration of either theanti-CSF1R antibody or immune stimulating agent alone.

In some embodiments, the at least one immune stimulating agent comprisesan antagonist of an inhibitor of the activation of T cells, while insome embodiments, the at least one immune stimulating agent comprisescomprises an agonist of a stimulator of the activation of T cells. Insome embodiments, the at least one immune stimulating agent comprises anantagonist of CTLA4, LAG-3, Galectin 1, Galectin 9, CEACAM-1, BTLA,CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP,PD1H, LAIR1, TIM1, TIM3, TIM4, ILT4, IL-6, IL-10, TGFβ, VEGF, KIR,LAG-3, adenosine A2A receptor, PI3Kdelta, or IDO. In some embodiments,the at least one immune stimulating agent comprises an agonist of B7-1,B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR,GITRL, CD27, CD40, CD40L, DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21,IFNα, STING or a Toll-like receptor agonist such as a TLR2/4 agonist. Insome embodiments, the at least one immune stimulating agent comprises anagent that binds to a member of the B7 family of membrane-bound proteinssuch as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), andB7-H6. In some embodiments, the at least one immune stimulating agentcomprises an agent that binds to a member of the TNF receptor family ora co-stimulatory or co-inhibitory molecule binding to a member of theTNF receptor family such as CD40, CD40L, OX40, OX40L, GITR, GITRL, CD70,CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4,TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK,BAFFR, EDAR, XEDAR, EDA1, EDA2, TACI, APRIL, BCMA, LTβR, LIGHT, DeR3,HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFβ, TNFR2, TNFα, 1β2, FAS, FASL,RELT, DR6, TROY, or NGFβ. In some embodiments, the at least one immunestimulating agent comprises an agent that antagonizes or inhibits acytokine that inhibits T cell activation such as IL-6, IL-10, TGFβ,VEGF. In some embodiments, the at least one immune stimulating agentcomprises an antagonist of a chemokine, such as CXCR2, CXCR4, CCR2, orCCR4. In some embodiments, the at least one immune stimulating agentcomprises an agonist of a cytokine that stimulates T cell activationsuch as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα. In some embodiments,the at least one immune stimulating agent comprises an antibody. In someembodiments, the at least one immune stimulating agent may comprise avaccine, such as a mesothelin-targeting vaccine or attenuated listeriacancer vaccine such as CRS-207. Any one or more of the aboveantagonists, agonists, and binding agents may be combined with any oneor more of the anti-CSF1R antibodies described herein.

In some embodiments, the at least one immune stimulating agent comprisesa CD40 agonist, optionally in combination with at least one other immunestimulating agent as listed above. In some embodiments, the CD40 agonistis an antibody. In some embodiments, the CD40 agonist is an anti-CD40antibody. In some embodiments, the anti-CD40 antibody comprises the CDRsof an antibody selected from CP-870,893; dacetuzumab; SEA-CD40;ADC-1013; RO7009789; and Chi Lob 7/4. In some embodiments, the anti-CD40antibody comprises the heavy chain and light chain variable regions ofan antibody selected from CP-870,893; dacetuzumab; SEA-CD40; ADC-1013;RO7009789; and Chi Lob 7/4. In some embodiments, the anti-CD40 antibodyis an antibody selected from CP-870,893; dacetuzumab; SEA-CD40;ADC-1013; RO7009789; and Chi Lob 7/4. In some embodiments, the CD40agonist is recombinant CD40L. In some embodiments, the at least oneimmune stimulating agent comprises a CD40 agonist and at least oneadditional immune stimulating agent from any of those described above.For example, any one or more of the above immune stimulating agentsabove may be combined with any one or more of the anti-CSF1R antibodiesdescribed herein as well as with a CD40 agonist, such as a CD40 agonistantibody or recombinant CD40L, such as any one of the anti-CD40antibodies described above.

In some embodiments, the anti-CSF1R antibody and the at least one immunestimulatory agent are administered concurrently or sequentially. In someembodiments, the anti-CSF1R antibody and the at least one immunestimulatory agent are administered concurrently. In some embodiments,one or more doses of the at least one immune stimulatory agent areadministered prior to administering an anti-CSF1R antibody. In someembodiments, the subject received a complete course of therapy with theat least one immune stimulatory agent prior to administration of theanti-CSF1R antibody. In some embodiments, the anti-CSF1R antibody isadministered during a second course of therapy with the at least oneimmune stimulatory agent. In some embodiments, the subject received atleast one, at least two, at least three, or at least four doses of theat least one immune stimulatory agent prior to administration of theanti-CSF1R antibody. In some embodiments, at least one dose of the atleast one immune stimulatory agent is administered concurrently with theanti-CSF1R inhibitor. In some embodiments, one or more doses of theanti-CSF1R antibody are administered prior to administering at least oneimmune stimulatory agent. In some embodiments, the subject received atleast two, at least three, or at least four doses of the anti-CSF1Rantibody prior to administration of at least one immune stimulatoryagent. In some embodiments, at least one dose of the anti-CSF1R antibodyis administered concurrently with the at least one immune stimulatoryagent.

In some embodiments, the cancer is selected from non-small cell lungcancer, melanoma, squamous cell carcinoma of the head and neck, ovariancancer, pancreatic cancer, renal cell carcinoma, hepatocellularcarcinoma, bladder cancer, endometrial cancer, Hodgkin's lymphoma, lungcancer, glioma, gioblastoma multiforme, colon cancer, breast cancer,bone cancer, skin cancer, uterine cancer, gastric cancer, stomachcancer, lymphoma, lymphocytic leukemia, multiple myeloma, prostatecancer, mesothelioma, and kidney cancer. In some embodiments, the canceris recurrent or progressive after a therapy selected from surgery,chemotherapy, radiation therapy, or a combination thereof.

In some embodiments, compositions are provided, comprising an anti-CSF1Rantibody and at least one immune stimulatory agent. In some embodiments,the at least one immune stimulating agent comprises an antagonist of aninhibitor of the activation of T cells, while in some embodiments, theat least one immune stimulating agent comprises comprises an agonist ofa stimulator of the activation of T cells. In some embodiments, the atleast one immune stimulating agent comprises an antagonist of CTLA4,LAG-3, Galectin 1, Galectin 9, CEACAM-1, BTLA, CD25, CD69, TIGIT, CD113,GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM1, TIM3,TIM4, ILT4, IL-6, IL-10, TGFβ, VEGF, KIR, LAG-3, adenosine A2A receptor,PI3Kdelta, or IDO. In some embodiments, the at least one immunestimulating agent comprises an agonist of B7-1, B7-2, CD28, 4-1BB(CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD27, CD40,CD40L, DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21, IFNα, STING, or aToll-like receptor agonist such as a TLR2/4 agonist. In someembodiments, the at least one immune stimulating agent comprises anagent that binds to a member of the B7 family of membrane-bound proteinssuch as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), andB7-H6. In some embodiments, the at least one immune stimulating agentcomprises an agent that binds to a member of the TNF receptor family ora co-stimulatory or co-inhibitory molecule binding to a member of theTNF receptor family such as CD40, CD40L, OX40, OX40L, GITR, GITRL, CD70,CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4,TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK,BAFFR, EDAR, XEDAR, EDA1, EDA2, TACI, APRIL, BCMA, LTβR, LIGHT, DeR3,HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFβ, TNFR2, TNFα, 1β2, FAS, FASL,RELT, DR6, TROY, or NGFβ. In some embodiments, the at least one immunestimulating agent comprises an agent that antagonizes or inhibits acytokine that inhibits T cell activation such as IL-6, IL-10, TGFβ,VEGF. In some embodiments, the at least one immune stimulating agentcomprises an agonist of a cytokine that stimulates T cell activationsuch as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα. In some embodiments,the at least one immune stimulating agent comprises an antagonist of achemokine, such as CXCR2, CXCR4, CCR2, or CCR4. In some embodiments, theat least one immune stimulating agent comprises an antibody. In someembodiments, the at least one immune stimulating agent may comprise avaccine, such as a mesothelin-targeting vaccine or attenuated listeriacancer vaccine such as CRS-207.

In some embodiments, the compositions comprise any one or more of theabove antagonists, agonists, and binding agents combined with any one ormore of the anti-CSF1R antibodies described herein. The compositions mayinclude each therapeutic agent in a separate container or compartment oralternatively, may include two or more of the therapeutic agents mixedtogether.

In some embodiments, the compositions comprise an anti-CSF1R antibodyand a CD40 agonist, optionally along with at least one other immunestimulating agent as listed above. In some embodiments, the CD40 agonistis an anti-CD40 antibody. In some embodiments, the anti-CD40 antibodycomprises the CDRs of an antibody selected from CP-870,893; dacetuzumab;SEA-CD40; ADC-1013; RO7009789; and Chi Lob 7/4. In some embodiments, theanti-CD40 antibody comprises the heavy chain and light chain variableregions of an antibody selected from CP-870,893; dacetuzumab; SEA-CD40;ADC-1013; RO7009789; and Chi Lob 7/4. In some embodiments, the anti-CD40antibody is an antibody selected from CP-870,893; dacetuzumab; SEA-CD40;ADC-1013; RO7009789; and Chi Lob 7/4. In some embodiments, the CD40agonist is recombinant CD40L. In some embodiments, the compositionscomprise any one or more of the above immune stimulating agents abovecombined with both any one or more of the anti-CSF1R antibodiesdescribed herein as well as with a CD40 agonist, such as a CD40 agonistantibody or recombinant CD40L, such as any one of the anti-CD40antibodies described above. The compositions may include eachtherapeutic agent in a separate container or compartment oralternatively, may include two or more of the therapeutic agents mixedtogether.

In any of the compositions or methods described herein, the antibodyheavy chain and/or the antibody light chain of the anti-CSF1R antibodymay have the structure described below.

In any of the compositions or methods described herein, the anti-CSF1Rantibody heavy chain may comprise a sequence that is at least 90%, atleast 95%, at least 97%, at least 99%, or 100% identical to a sequenceselected from SEQ ID NOs: 9, 11, 13, and 39 to 45. In any of the methodsdescribed herein, the anti-CSF1R antibody light chain may comprise asequence that is at least 90%, at least 95%, at least 97%, at least 99%,or 100% identical to a sequence selected from SEQ ID NOs: 10, 12, 14,and 46 to 52. In any of the compositions or methods described herein,the anti-CSF1R antibody heavy chain may comprise a sequence that is atleast 90%, at least 95%, at least 97%, at least 99%, or 100% identicalto a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45, and theanti-CSF1R antibody light chain may comprise a sequence that is at least90%, at least 95%, at least 97%, at least 99%, or 100% identical to asequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52.

In any of the compositions or methods described herein, the anti-CSF1Rantibody HC CDR1, HC CDR2, and HC CDR3 may comprise a set of sequencesselected from: (a) SEQ ID NOs: 15, 16, and 17; (b) SEQ ID NOs: 21, 22,and 23; and (c) SEQ ID NOs: 27, 28, and 29. In any of the compositionsor methods described herein, the anti-CSF1R antibody LC CDR1, LC CDR2,and LC CDR3 may comprise a set of sequences selected from: (a) SEQ IDNOs: 18, 19, and 20; (b) SEQ ID NOs: 24, 25, and 26; and (c) SEQ ID NOs:30, 31, and 32.

In any of the compositions or methods described herein, the anti-CSF1Rantibody heavy chain may comprise an HC CDR1, HC CDR2, and HC CDR3,wherein the HC CDR1, HC CDR2, and HC CDR3 comprise a set of sequencesselected from: (a) SEQ ID NOs: 15, 16, and 17; (b) SEQ ID NOs: 21, 22,and 23; and (c) SEQ ID NOs: 27, 28, and 29; and the light chain maycomprise an LC CDR1, LC CDR2, and LC CDR3, wherein the LC CDR1, LC CDR2,and LC CDR3 comprise a set of sequences selected from: (a) SEQ ID NOs:18, 19, and 20; (b) SEQ ID NOs: 24, 25, and 26; and (c) SEQ ID NOs: 30,31, and 32.

In any of the compositions or methods described herein, the anti-CSF1Rantibody may comprise: (a) a heavy chain comprising a sequence that isat least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 9 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 10; (b) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 11 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 12; (c) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 13 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 14; (d) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 39 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 46; (e) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 40 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 46; (f) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 41 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 46; (g) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 39 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 47; (h) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 40 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 47; (i) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 41 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 47; and (j) aheavy chain comprising a sequence that is at least 95%, at least 97%, atleast 99%, or 100% identical to SEQ ID NO: 42 and a light chaincomprising a sequence that is at least 95%, at least 97%, at least 99%,or 100% identical to SEQ ID NO: 48; (k) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 42 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 49; (1) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 42 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 50; (m) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 43 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 48; (n) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 43 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 49; (o) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 43 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 50; (p) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 44 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 51; (q) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 44 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 52; (r) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 45 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 51; or (s) a heavy chain comprisinga sequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 45 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 52.

In any of the compositions or methods described herein, the anti-CSF1Rantibody may comprise: (a) a heavy chain comprising a heavy chain (HC)CDR1 having the sequence of SEQ ID NO: 15, an HC CDR2 having thesequence of SEQ ID NO: 16, and an HC CDR3 having the sequence of SEQ IDNO: 17, and a light chain comprising a light chain (LC) CDR1 having thesequence of SEQ ID NO: 18, a LC CDR2 having the sequence of SEQ ID NO:19, and a LC CDR3 having the sequence of SEQ ID NO: 20; (b) a heavychain comprising a heavy chain (HC) CDR1 having the sequence of SEQ IDNO: 21, an HC CDR2 having the sequence of SEQ ID NO: 22, and an HC CDR3having the sequence of SEQ ID NO: 23, and a light chain comprising alight chain (LC) CDR1 having the sequence of SEQ ID NO: 24, a LC CDR2having the sequence of SEQ ID NO: 25, and a LC CDR3 having the sequenceof SEQ ID NO: 26; or (c) a heavy chain comprising a heavy chain (HC)CDR1 having the sequence of SEQ ID NO: 27, an HC CDR2 having thesequence of SEQ ID NO: 28, and an HC CDR3 having the sequence of SEQ IDNO: 29, and a light chain comprising a light chain (LC) CDR1 having thesequence of SEQ ID NO: 30, a LC CDR2 having the sequence of SEQ ID NO:31, and a LC CDR3 having the sequence of SEQ ID NO: 32.

In any of the compositions or methods described herein, the anti-CSF1Rantibody may comprise: (a) a heavy chain comprising a sequence of SEQ IDNO: 53 and a light chain comprising a sequence of SEQ ID NO: 60; (b) aheavy chain comprising a sequence of SEQ ID NO: 53 and a light chaincomprising a sequence of SEQ ID NO: 61; or (c) a heavy chain comprisinga sequence of SEQ ID NO: 58 and a light chain comprising a sequence ofSEQ ID NO: 65. In some embodiments, an antibody comprises a heavy chainand a light chain, wherein the antibody comprises: (a) a heavy chainconsisting of the sequence of SEQ ID NO: 53 and a light chain consistingof the sequence of SEQ ID NO: 60; (b) a heavy chain consisting of thesequence of SEQ ID NO: 53 and a light chain consisting of the sequenceof SEQ ID NO: 61; or (c) a heavy chain consisting of the sequence of SEQID NO: 58 and a light chain consisting of the sequence of SEQ ID NO: 65.

In any of the compositions or methods described herein, the anti-CSF1Rantibody may be a humanized antibody. In any of the compositions ormethods described herein, the anti-CSF1R antibody may be selected from aFab, an Fv, an scFv, a Fab′, and a (Fab′)₂. In any of the compositionsor methods described herein, the anti-CSF1R antibody may be a chimericantibody. In any of the compositions or methods described herein, theanti-CSF1R antibody may be selected from an IgA, an IgG, and an IgD. Inany of the compositions or methods described herein, the anti-CSF1Rantibody may be an IgG. In any of the methods described herein, theantibody may be an IgG1 or IgG2.

In any of the compositions or methods described herein, the anti-CSF1Rantibody may bind to human CSF1R and/or binds to cynomolgus CSF1R. Inany of the compositions or methods described herein, the anti-CSF1Rantibody may block ligand binding to CSF1R. In any of the compositionsor methods described herein, the anti-CSF1R antibody may block bindingof CSF1 and/or IL-34 to CSF1R. In any of the compositions or methodsdescribed herein, the anti-CSF1R antibody may block binding of both CSF1and IL-34 to CSF1R. In any of the compositions or methods describedherein, the anti-CSF1R antibody may inhibit ligand-induced CSF1Rphosphorylation. In any of the compositions or methods described herein,the anti-CSF1R antibody may inhibit CSF1- and/or IL-34-induced CSF1Rphosphorylation. In any of the compositions or methods described herein,the anti-CSF1R antibody may bind to human CSF1R with an affinity (K_(D))of less than 1 nM. In any of the compositions or methods describedherein, the anti-CSF1R antibody may inhibit monocyte proliferationand/or survival responses in the presence of CSF1 or IL-34.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C show an alignment of the humanized heavy chain variableregions for each of humanized antibodies huAb1 to huAb16, as discussedin Example 1. Boxed residues are amino acids in the human acceptorsequence that were changed back to the corresponding mouse residue.

FIG. 2A-C show an alignment of the humanized light chain variableregions for each of humanized antibodies huAb1 to huAb16, as discussedin Example 1. Boxed amino acids are residues in the human acceptorsequence that were changed back to the corresponding mouse residue.

FIG. 3 shows that the combination of anti-CSF1R antibody and anti-CD40antibody demonstrate greater tumor growth suppression in an MC38 tumormouse model than either therapy alone.

FIG. 4A-B shows the tumor volume of individual mice at (FIG. 4A) day 11and (FIG. 4B) day 13. The combination of anti-CSF1R antibody andanti-CD40 antibody performed significantly better than either therapyalone at both time points.

FIG. 5 shows body weight in mice used in the study.

DETAILED DESCRIPTION

Tumor-associated macrophages (TAMs) are implicated in the pathogenesisof many cancers, and correlate with poor prognosis. TAMs can suppress ananti-tumor responses through multiple mechanisms. TAMs expressanti-inflammatory cytokines such as TGFβ and IL-10 which acts tosuppress the ability of intratumoral dendritic cells to stimulatecytotoxic T cell responses (Ruffell et al., 2014, Cancer Cell). TAMsalso express chemokines which recruit immunosuppressive regulatory Tcells into tumors (Curiel et al., 2004, Nature Med.; Mizukami et al.,2008, Int. J. Cancer), Moreover, TAMs express the ligands for the T cellinhibitor receptors PD-1 and CTLA-4 which act to directly inhibit T cellactivation and function. Inhibition of CSF1R can reduceimmunosuppressive TAMs in mouse models and human tumors. See, e.g., Rieset al., 2014, Cancer Cell, 25: 846-859; Pyontech et al., 2013, NatureMed., 19: 1264-1272; and Zhu et al., 2014, Cancer Res., 74: 5057-5069.In contrast to CSF1R blockade which acts by reducing immunosuppression,immune stimulating agents work by stimulating an immune response.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Exemplary techniques used in connection with recombinant DNA,oligonucleotide synthesis, tissue culture and transformation (e.g.,electroporation, lipofection), enzymatic reactions, and purificationtechniques are known in the art. Many such techniques and procedures aredescribed, e.g., in Sambrook et al. Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)), among other places. In addition, exemplarytechniques for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients arealso known in the art.

In this application, the use of “or” means “and/or” unless statedotherwise. In the context of a multiple dependent claim, the use of “or”refers back to more than one preceding independent or dependent claim inthe alternative only. Also, terms such as “element” or “component”encompass both elements and components comprising one unit and elementsand components that comprise more than one subunit unless specificallystated otherwise.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The terms “nucleic acid molecule” and “polynucleotide” may be usedinterchangeably, and refer to a polymer of nucleotides. Such polymers ofnucleotides may contain natural and/or non-natural nucleotides, andinclude, but are not limited to, DNA, RNA, and PNA. “Nucleic acidsequence” refers to the linear sequence of nucleotides that comprise thenucleic acid molecule or polynucleotide.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

The term “CSF1R” refers herein to the full-length CSF1R, which includesthe N-terminal ECD, the transmembrane domain, and the intracellulartyrosine kinase domain, with or without an N-terminal leader sequence.In some embodiments, the CSF1R is a human CSF1R having the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The term “immune stimulating agent” as used herein refers to a moleculethat stimulates the immune system by either acting as an agonist of animmune-stimulatory molecule, including a co-stimulatory molecule, oracting as an antagonist of an immune inhibitory molecule, including aco-inhibitory molecule. An immune stimulating agent may be a biologic,such as an antibody or antibody fragment, other protein, or vaccine, ormay be a small molecule drug. An “immune stimulatory molecule” includesa receptor or ligand that acts to enhance, stimulate, induce, orotherwise “turn-on” an immune response. Immune stimulatory molecules asdefined herein include co-stimulatory molecules. An “immune inhibitorymolecule” includes a receptor or ligand that acts to reduce, inhibit,suppress, or otherwise “turn-off” an immune response. Immune inhibitorymolecules as defined herein include co-inhibitory molecules. Such immunestimulatory and immune inhibitory molecules may be, for example,receptors or ligands found on immune cells such as a T cells, or foundon cells involved in innate immunity such as NK cells.

The terms “B-cell surface antigen CD40” and “CD40” refer herein to thefull-length CD40, which includes the N-terminal ECD, the transmembranedomain, and the intracellular domain, with or without an N-terminalleader sequence. In some embodiments, the CD40 is a human CD40 havingthe amino acid sequence of SEQ ID NO: 96 (precursor, with signalsequence) or SEQ ID NO: 97 (mature, without signal sequence).

The term “CD40 agonist” refers to a moiety that interacts with CD40 andenhances CD40 activity. Nonlimiting exemplary CD40 activities includesignaling through CD40, enhancement of antigen presenting activity,induction of proinflammatory cytokines, and induction of tumoricidalactivity. In some embodiments, a CD40 agonist is an anti-CD40 antibody.

The term “anti-CD40 antibody” refers to an antibody that specificallybinds to CD40. Unless specifically indicated otherwise, the term“anti-CD40 antibody” as used herein refers to an anti-CD40 agonistantibody.

With reference to anti-CSF1R antibodies the term “blocks binding of” aligand, such as CSF1 and/or IL-34, and grammatical variants thereof, areused to refer to the ability to inhibit the interaction between CSF1Rand a CSF1R ligand, such as CSF1 and/or IL-34. Such inhibition may occurthrough any mechanism, including direct interference with ligandbinding, e.g., because of overlapping binding sites on CSF1R, and/orconformational changes in CSF1R induced by the antibody that alterligand affinity, etc. Antibodies and antibody fragments referred to as“functional” are characterized by having such properties.

The term “antibody” as used herein refers to a molecule comprising atleast complementarity-determining region (CDR) 1, CDR2, and CDR3 of aheavy chain and at least CDR1, CDR2, and CDR3 of a light chain, whereinthe molecule is capable of binding to antigen. The term antibodyincludes, but is not limited to, fragments that are capable of bindingantigen, such as Fv, single-chain Fv (scFv), Fab, Fab′, and (Fab′)₂. Theterm antibody also includes, but is not limited to, chimeric antibodies,humanized antibodies, and antibodies of various species such as mouse,human, cynomolgus monkey, etc.

In some embodiments, an antibody comprises a heavy chain variable regionand a light chain variable region. In some embodiments, an antibodycomprises at least one heavy chain comprising a heavy chain variableregion and at least a portion of a heavy chain constant region, and atleast one light chain comprising a light chain variable region and atleast a portion of a light chain constant region. In some embodiments,an antibody comprises two heavy chains, wherein each heavy chaincomprises a heavy chain variable region and at least a portion of aheavy chain constant region, and two light chains, wherein each lightchain comprises a light chain variable region and at least a portion ofa light chain constant region. As used herein, a single-chain Fv (scFv),or any other antibody that comprises, for example, a single polypeptidechain comprising all six CDRs (three heavy chain CDRs and three lightchain CDRs) is considered to have a heavy chain and a light chain. Insome such embodiments, the heavy chain is the region of the antibodythat comprises the three heavy chain CDRs and the light chain in theregion of the antibody that comprises the three light chain CDRs.

The term “heavy chain variable region” as used herein refers to a regioncomprising heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. Insome embodiments, a heavy chain variable region also comprises at leasta portion of an FR1 and/or at least a portion of an FR4. In someembodiments, a heavy chain CDR1 corresponds to Kabat residues 26 to 35;a heavy chain CDR2 corresponds to Kabat residues 50 to 65; and a heavychain CDR3 corresponds to Kabat residues 95 to 102. See, e.g., KabatSequences of Proteins of Immunological Interest (1987 and 1991, NIH,Bethesda, Md.); and FIG. 1 . In some embodiments, a heavy chain CDR1corresponds to Kabat residues 31 to 35; a heavy chain CDR2 correspondsto Kabat residues 50 to 65; and a heavy chain CDR3 corresponds to Kabatresidues 95 to 102. See id.

The term “heavy chain constant region” as used herein refers to a regioncomprising at least three heavy chain constant domains, C_(H)1, C_(H)2,and C_(H)3. Nonlimiting exemplary heavy chain constant regions includeγ,δ, and α. Nonlimiting exemplary heavy chain constant regions alsoinclude ε and μ. Each heavy constant region corresponds to an antibodyisotype. For example, an antibody comprising a γ constant region is anIgG antibody, an antibody comprising a δ constant region is an IgDantibody, and an antibody comprising an α constant region is an IgAantibody. Further, an antibody comprising a μ constant region is an IgMantibody, and an antibody comprising an ε constant region is an IgEantibody. Certain isotypes can be further subdivided into subclasses.For example, IgG antibodies include, but are not limited to, IgG1(comprising a γ₁ constant region), IgG2 (comprising a γ₂ constantregion), IgG3 (comprising a γ₃ constant region), and IgG4 (comprising aγ₄ constant region) antibodies; IgA antibodies include, but are notlimited to, IgA1 (comprising an α₁ constant region) and IgA2 (comprisingan α₂ constant region) antibodies; and IgM antibodies include, but arenot limited to, IgM1 and IgM2.

In some embodiments, a heavy chain constant region comprises one or moremutations (or substitutions), additions, or deletions that confer adesired characteristic on the antibody. A nonlimiting exemplary mutationis the S241P mutation in the IgG4 hinge region (between constant domainsC_(H)1 and C_(H)2), which alters the IgG4 motif CPSCP to CPPCP, which issimilar to the corresponding motif in IgG1. That mutation, in someembodiments, results in a more stable IgG4 antibody. See, e.g., Angal etal., Mol. Immunol. 30: 105-108 (1993); Bloom et al., Prot. Sci. 6:407-415 (1997); Schuurman et al., Mol. Immunol. 38: 1-8 (2001).

The term “heavy chain” as used herein refers to a polypeptide comprisingat least a heavy chain variable region, with or without a leadersequence. In some embodiments, a heavy chain comprises at least aportion of a heavy chain constant region. The term “full-length heavychain” as used herein refers to a polypeptide comprising a heavy chainvariable region and a heavy chain constant region, with or without aleader sequence.

The term “light chain variable region” as used herein refers to a regioncomprising light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. Insome embodiments, a light chain variable region also comprises an FR1and/or an FR4. In some embodiments, a light chain CDR1 corresponds toKabat residues 24 to 34; a light chain CDR2 corresponds to Kabatresidues 50 to 56; and a light chain CDR3 corresponds to Kabat residues89 to 97. See, e.g., Kabat Sequences of Proteins of ImmunologicalInterest (1987 and 1991, NIH, Bethesda, Md.); and FIG. 1 .

The term “light chain constant region” as used herein refers to a regioncomprising a light chain constant domain, CL. Nonlimiting exemplarylight chain constant regions include λ, and κ.

The term “light chain” as used herein refers to a polypeptide comprisingat least a light chain variable region, with or without a leadersequence. In some embodiments, a light chain comprises at least aportion of a light chain constant region. The term “full-length lightchain” as used herein refers to a polypeptide comprising a light chainvariable region and a light chain constant region, with or without aleader sequence.

A “chimeric antibody” as used herein refers to an antibody comprising atleast one variable region from a first species (such as mouse, rat,cynomolgus monkey, etc.) and at least one constant region from a secondspecies (such as human, cynomolgus monkey, etc.). In some embodiments, achimeric antibody comprises at least one mouse variable region and atleast one human constant region. In some embodiments, a chimericantibody comprises at least one cynomolgus variable region and at leastone human constant region. In some embodiments, a chimeric antibodycomprises at least one rat variable region and at least one mouseconstant region. In some embodiments, all of the variable regions of achimeric antibody are from a first species and all of the constantregions of the chimeric antibody are from a second species.

A “humanized antibody” as used herein refers to an antibody in which atleast one amino acid in a framework region of a non-human variableregion has been replaced with the corresponding amino acid from a humanvariable region. In some embodiments, a humanized antibody comprises atleast one human constant region or fragment thereof. In someembodiments, a humanized antibody is a Fab, an scFv, a (Fab′)₂, etc.

A “CDR-grafted antibody” as used herein refers to a humanized antibodyin which the complementarity determining regions (CDRs) of a first(non-human) species have been grafted onto the framework regions (FRs)of a second (human) species.

A “human antibody” as used herein refers to antibodies produced inhumans, antibodies produced in non-human animals that comprise humanimmunoglobulin genes, such as XenoMouse®, and antibodies selected usingin vitro methods, such as phage display, wherein the antibody repertoireis based on a human immunoglobulin sequences.

The term “leader sequence” refers to a sequence of amino acid residueslocated at the N terminus of a polypeptide that facilitates secretion ofa polypeptide from a mammalian cell. A leader sequence may be cleavedupon export of the polypeptide from the mammalian cell, forming a matureprotein. Leader sequences may be natural or synthetic, and they may beheterologous or homologous to the protein to which they are attached.Exemplary leader sequences include, but are not limited to, antibodyleader sequences, such as, for example, the amino acid sequences of SEQID NOs: 3 and 4, which correspond to human light and heavy chain leadersequences, respectively. Nonlimiting exemplary leader sequences alsoinclude leader sequences from heterologous proteins. In someembodiments, an antibody lacks a leader sequence. In some embodiments,an antibody comprises at least one leader sequence, which may beselected from native antibody leader sequences and heterologous leadersequences.

The term “vector” is used to describe a polynucleotide that may beengineered to contain a cloned polynucleotide or polynucleotides thatmay be propagated in a host cell. A vector may include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that may be used in colorimetric assays,e.g., β-galactosidase). The term “expression vector” refers to a vectorthat is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NSO cells, PER.C6® cells(Crucell), and 293 and CHO cells, and their derivatives, such as 293-6Eand DG44 cells, respectively.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature. For example, a polypeptide is referred to as“isolated” when it is separated from at least some of the components ofthe cell in which it was produced. Where a polypeptide is secreted by acell after expression, physically separating the supernatant containingthe polypeptide from the cell that produced it is considered to be“isolating” the polypeptide. Similarly, a polynucleotide is referred toas “isolated” when it is not part of the larger polynucleotide (such as,for example, genomic DNA or mitochondrial DNA, in the case of a DNApolynucleotide) in which it is typically found in nature, or isseparated from at least some of the components of the cell in which itwas produced, e.g., in the case of an RNA polynucleotide. Thus, a DNApolynucleotide that is contained in a vector inside a host cell may bereferred to as “isolated” so long as that polynucleotide is not found inthat vector in nature.

The term “elevated level” means a higher level of a protein in aparticular tissue of a subject relative to the same tissue in a control,such as an individual or individuals who are not suffering from canceror other condition described herein. The elevated level may be theresult of any mechanism, such as increased expression, increasedstability, decreased degradation, increased secretion, decreasedclearance, etc., of the protein.

The term “reduce” or “reduces” means to lower the level of a protein ina particular tissue of a subject by at least 10%. In some embodiments,an agent, such as an antibody that binds CSF1R, reduces the level of aprotein in a particular tissue of a subject by at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, or at least 90%. In someembodiments, the level of a protein is reduced relative to the level ofthe protein prior to contacting with an agent, such as an antibody thatbinds CSF1R.

The term “resistant,” when used in the context of resistance to atherapeutic agent, means a decreased response or lack of response to astandard dose of the therapeutic agent, relative to the subject'sresponse to the standard dose of the therapeutic agent in the past, orrelative to the expected response of a similar subject with a similardisorder to the standard dose of the therapeutic agent. Thus, in someembodiments, a subject may be resistant to therapeutic agent althoughthe subject has not previously been given the therapeutic agent, or thesubject may develop resistance to the therapeutic agent after havingresponded to the agent on one or more previous occasions.

The terms “subject” and “patient” are used interchangeably herein torefer to a human. In some embodiments, methods of treating othermammals, including, but not limited to, rodents, simians, felines,canines, equines, bovines, porcines, ovines, caprines, mammalianlaboratory animals, mammalian farm animals, mammalian sport animals, andmammalian pets, are also provided.

The term “sample,” as used herein, refers to a composition that isobtained or derived from a subject that contains a cellular and/or othermolecular entity that is to be characterized, quantitated, and/oridentified, for example based on physical, biochemical, chemical and/orphysiological characteristics. An exemplary sample is a tissue sample.

The term “tissue sample” refers to a collection of similar cellsobtained from a tissue of a subject. The source of the tissue sample maybe solid tissue as from a fresh, frozen and/or preserved organ or tissuesample or biopsy or aspirate; blood or any blood constituents; bodilyfluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid,synovial fluid, or interstitial fluid; cells from any time in gestationor development of the subject. In some embodiments, a tissue sample is asynovial biopsy tissue sample and/or a synovial fluid sample. In someembodiments, a tissue sample is a synovial fluid sample. The tissuesample may also be primary or cultured cells or cell lines. Optionally,the tissue sample is obtained from a disease tissue/organ. The tissuesample may contain compounds that are not naturally intermixed with thetissue in nature such as preservatives, anticoagulants, buffers,fixatives, nutrients, antibiotics, or the like. A “control sample” or“control tissue”, as used herein, refers to a sample, cell, or tissueobtained from a source known, or believed, not to be afflicted with thedisease for which the subject is being treated.

For the purposes herein a “section” of a tissue sample means a part orpiece of a tissue sample, such as a thin slice of tissue or cells cutfrom a solid tissue sample.

The term “cancer” is used herein to refer to a group of cells thatexhibit abnormally high levels of proliferation and growth. A cancer maybe benign (also referred to as a benign tumor), pre-malignant, ormalignant. Cancer cells may be solid cancer cells or leukemic cancercells. The term “cancer growth” is used herein to refer to proliferationor growth by a cell or cells that comprise a cancer that leads to acorresponding increase in the size or extent of the cancer.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular nonlimiting examples ofsuch cancers include squamous cell cancer, small-cell lung cancer,pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma,non-small cell lung cancer (including squamous cell non-small cell lungcancer), adenocarcinoma of the lung, squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney cancer, renal cell carcinoma, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, braincancer, endometrial cancer, testis cancer, cholangiocarcinoma,gallbladder carcinoma, gastric cancer, melanoma, and various types ofhead and neck cancer (including squamous cell carcinoma of the head andneck).

The term “recurrent cancer” refers to a cancer that has returned after aprevious treatment regimen, following which there was a period of timeduring which the cancer could not be detected.

The term “progressive cancer” is a cancer that has increased in size ortumor spread since the beginning of a treatment regimen. In certainembodiments, a progressive cancer is a cancer that has increased in sizeor tumor spread by at least 10%, at least 20%, at least 30%, at least40%, or at least 50% since the beginning of a treatment regimen.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include, butare not limited to, alkylating agents such as thiotepa and Cytoxan®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., Taxol®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), Abraxane®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTaxotere® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;Gemzar® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; Navelbine® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g.,erlotinib (Tarceve®)) and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Further nonlimiting exemplary chemotherapeutic agents includeanti-hormonal agents that act to regulate or inhibit hormone action oncancers such as anti-estrogens and selective estrogen receptormodulators (SERMs), including, for example, tamoxifen (includingNolvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and Fareston® toremifene;aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, Megase® megestrol acetate, Aromasin®exemestane, formestanie, fadrozole, Rivisor® vorozole, Femara®letrozole, and Arimidex® anastrozole; and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as wellas troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in abherant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGFexpression inhibitor (e.g., Angiozyme® ribozyme) and a HER2 expressioninhibitor; vaccines such as gene therapy vaccines, for example,Allovectin® vaccine, Leuvectin® vaccine, and Vaxid® vaccine; Proleukin®rIL-2; Lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide (including, e.g., aninhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, arecombinant protein, an antibody, or conjugates or fusion proteinsthereof, that inhibits angiogenesis, vasculogenesis, or undesirablevascular permeability, either directly or indirectly. It should beunderstood that the anti-angiogenesis agent includes those agents thatbind and block the angiogenic activity of the angiogenic factor or itsreceptor. For example, an anti-angiogenesis agent is an antibody orother antagonist to an angiogenic agent, e.g., antibodies to VEGF-A(e.g., bevacizumab (Avastin®)) or to the VEGF-A receptor (e.g., KDRreceptor or Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec®(Imatinib Mesylate), small molecules that block VEGF receptor signaling(e.g., PTK787/ZK2284, SU6668, Sutent®/SU11248 (sunitinib malate),AMG706, or those described in, e.g., international patent application WO2004/113304). Anti-angiogensis agents also include native angiogenesisinhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun andD'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003)Oncogene 22:3172-3179 (e.g., Table 3 listing anti-angiogenic therapy inmalignant melanoma); Ferrara & Alitalo (1999) Nature Medicine5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table2 listing known anti-angiogenic factors); and, Sato (2003) Int. J. Clin.Oncol. 8:200-206 (e.g., Table 1 listing anti-angiogenic agents used inclinical trials).

A “growth inhibitory agent” as used herein refers to a compound orcomposition that inhibits growth of a cell (such as a cell expressingVEGF) either in vitro or in vivo. Thus, the growth inhibitory agent maybe one that significantly reduces the percentage of cells (such as acell expressing VEGF) in S phase. Examples of growth inhibitory agentsinclude, but are not limited to, agents that block cell cycleprogression (at a place other than S phase), such as agents that induceG1 arrest and M-phase arrest. Classical M-phase blockers include thevincas (vincristine and vinblastine), taxanes, and topoisomerase IIinhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, andbleomycin. Those agents that arrest G1 also spill over into S-phasearrest, for example, DNA alkylating agents such as tamoxifen,prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate,5-fluorouracil, and ara-C. Further information can be found inMendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1,entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” byMurakami et al. (W.B. Saunders, Philadelphia, 1995), e.g., p. 13. Thetaxanes (paclitaxel and docetaxel) are anticancer drugs both derivedfrom the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derivedfrom the European yew, is a semisynthetic analogue of paclitaxel(Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote theassembly of microtubules from tubulin dimers and stabilize microtubulesby preventing depolymerization, which results in the inhibition ofmitosis in cells.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent.Examples of therapeutic agents include, but are not limited to, e.g.,chemotherapeutic agents, growth inhibitory agents, cytotoxic agents,agents used in radiation therapy, anti-angiogenesis agents, cancerimmunotherapeutic agents, apoptotic agents, anti-tubulin agents, andother-agents to treat cancer, such as anti-HER-2 antibodies, anti-CD20antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g.,a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g.,erlotinib)(Tarceva®), platelet derived growth factor inhibitors (e.g.,Gleevec® (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib),interferons, CTLA4 inhibitors (e.g., anti-CTLA antibody ipilimumab(YERVOY®)), TIM3 inhibitors (e.g., anti-TIM3 antibodies), cytokines,antagonists (e.g., neutralizing antibodies) that bind to one or more ofthe following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL,BCMA, CTLA4, TIM3, or VEGF receptor(s), TRAIL/Apo2, and other bioactiveand organic chemical agents, etc. Combinations thereof are also includedin the invention.

An agent “antagonizes” factor activity when the agent neutralizes,blocks, inhibits, abrogates, reduces, and/or interferes with theactivity of the factor, including its binding to one or more receptorswhen the factor is a ligand.

“Treatment,” as used herein, refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathologic condition or disorder. Incertain embodiments, the term “treatment” covers any administration orapplication of a therapeutic for disease in a mammal, including a human,and includes inhibiting or slowing the disease or progression of thedisease; partially or fully relieving the disease, for example, bycausing regression, or restoring or repairing a lost, missing, ordefective function; stimulating an inefficient process; or causing thedisease plateau to have reduced severity. The term “treatment” alsoincludes reducing the severity of any phenotypic characteristic and/orreducing the incidence, degree, or likelihood of that characteristic.Those in need of treatment include those already with the disorder aswell as those prone to have the disorder or those in whom the disorderis to be prevented.

The term “effective amount” or “therapeutically effective amount” refersto an amount of a drug effective to treat a disease or disorder in asubject. In certain embodiments, an effective amount refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic or prophylactic result. A therapeutically effectiveamount of an anti-CSF1R antibody and/or an immune stimulating agent ofthe invention may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the antibodyor antibodies to elicit a desired response in the individual. Atherapeutically effective amount encompasses an amount in which anytoxic or detrimental effects of the antibody or antibodies areoutweighed by the therapeutically beneficial effects. In someembodiments, the expression “effective amount” refers to an amount ofthe antibody that is effective for treating the cancer.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, but not necessarily, since aprophylactic dose is used in subjects prior to or at an earlier stage ofdisease, the prophylactically effective amount would be less than thetherapeutically effective amount.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive (sequential)administration in any order.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed. For example, if the therapeutic agent is to be administeredorally, the carrier may be a gel capsule. If the therapeutic agent is tobe administered subcutaneously, the carrier ideally is not irritable tothe skin and does not cause injection site reaction.

Anti-CSF1R Antibodies

Anti-CSF1R antibodies include, but are not limited to, humanizedantibodies, chimeric antibodies, mouse antibodies, human antibodies, andantibodies comprising the heavy chain and/or light chain CDRs discussedherein.

Exemplary Humanized Antibodies

In some embodiments, humanized antibodies that bind CSF1R are provided.Humanized antibodies are useful as therapeutic molecules becausehumanized antibodies reduce or eliminate the human immune response tonon-human antibodies (such as the human anti-mouse antibody (HAMA)response), which can result in an immune response to an antibodytherapeutic, and decreased effectiveness of the therapeutic.

Nonlimiting exemplary humanized antibodies include huAb1 through huAb16,described herein. Nonlimiting exemplary humanized antibodies alsoinclude antibodies comprising a heavy chain variable region of anantibody selected from huAb1 to huAb16 and/or a light chain variableregion of an antibody selected from huAb1 to huAb16. Nonlimitingexemplary humanized antibodies include antibodies comprising a heavychain variable region selected from SEQ ID NOs: 39 to 45 and/or a lightchain variable region selected from SEQ ID NOs: 46 to 52. Exemplaryhumanized antibodies also include, but are not limited to, humanizedantibodies comprising heavy chain CDR1, CDR2, and CDR3, and/or lightchain CDR1, CDR2, and CDR3 of an antibody selected from 0301, 0302, and0311.

In some embodiments, a humanized anti-CSF1R antibody comprises heavychain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, and CDR3 ofan antibody selected from 0301, 0302, and 0311. Nonlimiting exemplaryhumanized anti-CSF1R antibodies include antibodies comprising sets ofheavy chain CDR1, CDR2, and CDR3 selected from: SEQ ID NOs: 15, 16, and17; SEQ ID NOs: 21, 22, and 23; and SEQ ID NOs: 27, 28, and 29.Nonlimiting exemplary humanized anti-CSF1R antibodies also includeantibodies comprising sets of light chain CDR1, CDR2, and CDR3 selectedfrom: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25, and 26; and SEQ IDNOs: 30, 31, and 32.

Nonlimiting exemplary humanized anti-CSF1R antibodies include antibodiescomprising the sets of heavy chain CDR1, CDR2, and CDR3, and light chainCDR1, CDR2, and CDR3 in Table 1 (SEQ ID NOs shown; see Table 8 forsequences). Each row of Table 1 shows the heavy chain CDR1, CDR2, andCDR3, and light chain CDR1, CDR2, and CDR3 of an exemplary antibody.

TABLE 1 Heavy chain and light chain CDRs Heavy chain Light chain CDR1CDR2 CDR3 CDR1 CDR2 CDR3 Ab SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID0301 15 16 17 18 19 20 0302 21 22 23 24 25 26 0311 27 28 29 30 31 32

Further Exemplary Humanized Antibodies

In some embodiments, a humanized anti-CSF1R antibody comprises a heavychain comprising a variable region sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45, and whereinthe antibody binds CSF1R. In some embodiments, a humanized anti-CSF1Rantibody comprises a light chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46to 52, wherein the antibody binds CSF1R. In some embodiments, ahumanized anti-CSF1R antibody comprises a heavy chain comprising avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 9, 11, 13, and 39 to 45; and a light chain comprising avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52; wherein the antibody binds CSF1R.

As used herein, whether a particular polypeptide is, for example, atleast 95% identical to an amino acid sequence can be determined using,e.g., a computer program. When determining whether a particular sequenceis, for example, 95% identical to a reference sequence, the percentageof identity is calculated over the full length of the reference aminoacid sequence.

In some embodiments, a humanized anti-CSF1R antibody comprises at leastone of the CDRs discussed herein. That is, in some embodiments, ahumanized anti-CSF1R antibody comprises at least one CDR selected from aheavy chain CDR1 discussed herein, a heavy chain CDR2 discussed herein,a heavy chain CDR3 discussed herein, a light chain CDR1 discussedherein, a light chain CDR2 discussed herein, and a light chain CDR3discussed herein. Further, in some embodiments, a humanized anti-CSF1Rantibody comprises at least one mutated CDR based on a CDR discussedherein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acidsubstitutions relative to the CDR discussed herein. In some embodiments,one or more of the amino acid substitutions are conservative amino acidsubstitutions. One skilled in the art can select one or more suitableconservative amino acid substitutions for a particular CDR sequence,wherein the suitable conservative amino acid substitutions are notpredicted to significantly alter the binding properties of the antibodycomprising the mutated CDR.

Exemplary humanized anti-CSF1R antibodies also include antibodies thatcompete for binding to CSF1R with an antibody described herein. Thus, insome embodiments, a humanized anti-CSF1R antibody is provided thatcompetes for binding to CSF1R with an antibody selected from Fabs 0301,0302, and 0311; and bivalent (i.e., having two heavy chains and twolight chains) antibody versions of those Fabs.

Exemplary Humanized Antibody Constant Regions

In some embodiments, a humanized antibody described herein comprises oneor more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from κ and λ. In some embodiments, a humanized antibodydescribed herein comprises a human IgG constant region. In someembodiments, a humanized antibody described herein comprises a humanIgG4 heavy chain constant region. In some such embodiments, a humanizedantibody described herein comprises an S241P mutation in the human IgG4constant region. In some embodiments, a humanized antibody describedherein comprises a human IgG4 constant region and a human κ light chain.

The choice of heavy chain constant region can determine whether or notan antibody will have effector function in vivo. Such effector function,in some embodiments, includes antibody-dependent cell-mediatedcytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), andcan result in killing of the cell to which the antibody is bound. Insome methods of treatment, including methods of treating some cancers,cell killing may be desirable, for example, when the antibody binds to acell that supports the maintenance or growth of the tumor. Exemplarycells that may support the maintenance or growth of a tumor include, butare not limited to, tumor cells themselves, cells that aid in therecruitment of vasculature to the tumor, and cells that provide ligands,growth factors, or counter-receptors that support or promote tumorgrowth or tumor survival. In some embodiments, when effector function isdesirable, an anti-CSF1R antibody comprising a human IgG1 heavy chain ora human IgG3 heavy chain is selected.

An antibody may be humanized by any method. Nonlimiting exemplarymethods of humanization include methods described, e.g., in U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; Jones etal., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-27(1988); Verhoeyen et al., Science 239: 1534-36 (1988); and U.S.Publication No. US 2009/0136500.

As noted above, a humanized antibody is an antibody in which at leastone amino acid in a framework region of a non-human variable region hasbeen replaced with the amino acid from the corresponding location in ahuman framework region. In some embodiments, at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least 10, at least 11, at least 12, atleast 15, or at least 20 amino acids in the framework regions of anon-human variable region are replaced with an amino acid from one ormore corresponding locations in one or more human framework regions.

In some embodiments, some of the corresponding human amino acids usedfor substitution are from the framework regions of different humanimmunoglobulin genes. That is, in some such embodiments, one or more ofthe non-human amino acids may be replaced with corresponding amino acidsfrom a human framework region of a first human antibody or encoded by afirst human immunoglobulin gene, one or more of the non-human aminoacids may be replaced with corresponding amino acids from a humanframework region of a second human antibody or encoded by a second humanimmunoglobulin gene, one or more of the non-human amino acids may bereplaced with corresponding amino acids from a human framework region ofa third human antibody or encoded by a third human immunoglobulin gene,etc. Further, in some embodiments, all of the corresponding human aminoacids being used for substitution in a single framework region, forexample, FR2, need not be from the same human framework. In someembodiments, however, all of the corresponding human amino acids beingused for substitution are from the same human antibody or encoded by thesame human immunoglobulin gene.

In some embodiments, an antibody is humanized by replacing one or moreentire framework regions with corresponding human framework regions. Insome embodiments, a human framework region is selected that has thehighest level of homology to the non-human framework region beingreplaced. In some embodiments, such a humanized antibody is aCDR-grafted antibody.

In some embodiments, following CDR-grafting, one or more framework aminoacids are changed back to the corresponding amino acid in a mouseframework region. Such “back mutations” are made, in some embodiments,to retain one or more mouse framework amino acids that appear tocontribute to the structure of one or more of the CDRs and/or that maybe involved in antigen contacts and/or appear to be involved in theoverall structural integrity of the antibody. In some embodiments, tenor fewer, nine or fewer, eight or fewer, seven or fewer, six or fewer,five or fewer, four or fewer, three or fewer, two or fewer, one, or zeroback mutations are made to the framework regions of an antibodyfollowing CDR grafting.

In some embodiments, a humanized antibody also comprises a human heavychain constant region and/or a human light chain constant region.

Exemplary Chimeric Antibodies

In some embodiments, an anti-CSF1R antibody is a chimeric antibody. Insome embodiments, an anti-CSF1R antibody comprises at least onenon-human variable region and at least one human constant region. Insome such embodiments, all of the variable regions of an anti-CSF1Rantibody are non-human variable regions, and all of the constant regionsof an anti-CSF1R antibody are human constant regions. In someembodiments, one or more variable regions of a chimeric antibody aremouse variable regions. The human constant region of a chimeric antibodyneed not be of the same isotype as the non-human constant region, ifany, it replaces. Chimeric antibodies are discussed, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851-55 (1984).

Nonlimiting exemplary chimeric antibodies include chimeric antibodiescomprising the heavy and/or light chain variable regions of an antibodyselected from 0301, 0302, and 0311. Additional nonlimiting exemplarychimeric antibodies include chimeric antibodies comprising heavy chainCDR1, CDR2, and CDR3, and/or light chain CDR1, CDR2, and CDR3 of anantibody selected from 0301, 0302, and 0311.

Nonlimiting exemplary chimeric anti-CSF1R antibodies include antibodiescomprising the following pairs of heavy and light chain variableregions: SEQ ID NOs: 9 and 10; SEQ ID NOs: 11 and 12; and SEQ ID NOs: 13and 14.

Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising a set of heavy chain CDR1, CDR2, and CDR3, and light chainCDR1, CDR2, and CDR3 shown above in Table 1.

Further Exemplary Chimeric Antibodies

In some embodiments, a chimeric anti-CSF1R antibody comprises a heavychain comprising a variable region sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45, wherein theantibody binds CSF1R. In some embodiments, a chimeric anti-CSF1Rantibody comprises a light chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46to 52, wherein the antibody binds CSF1R. In some embodiments, a chimericanti-CSF1R antibody comprises a heavy chain comprising a variable regionsequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical to a sequence selected from SEQ ID NOs: 9, 11,13, and 39 to 45; and a light chain comprising a variable regionsequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical to a sequence selected from SEQ ID NOs: 10, 12,14, and 46 to 52; wherein the antibody binds CSF1R.

In some embodiments, a chimeric anti-CSF1R antibody comprises at leastone of the CDRs discussed herein. That is, in some embodiments, achimeric anti-CSF1R antibody comprises at least one CDR selected from aheavy chain CDR1 discussed herein, a heavy chain CDR2 discussed herein,a heavy chain CDR3 discussed herein, a light chain CDR1 discussedherein, a light chain CDR2 discussed herein, and a light chain CDR3discussed herein. Further, in some embodiments, a chimeric anti-CSF1Rantibody comprises at least one mutated CDR based on a CDR discussedherein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acidsubstitutions relative to the CDR discussed herein. In some embodiments,one or more of the amino acid substitutions are conservative amino acidsubstitutions. One skilled in the art can select one or more suitableconservative amino acid substitutions for a particular CDR sequence,wherein the suitable conservative amino acid substitutions are notpredicted to significantly alter the binding properties of the antibodycomprising the mutated CDR.

Exemplary chimeric anti-CSF1R antibodies also include chimericantibodies that compete for binding to CSF1R with an antibody describedherein. Thus, in some embodiments, a chimeric anti-CSF1R antibody isprovided that competes for binding to CSF1R with an antibody selectedfrom Fabs 0301, 0302, and 0311; and bivalent (i.e., having two heavychains and two light chains) antibody versions of those Fabs.

Exemplary Chimeric Antibody Constant Regions

In some embodiments, a chimeric antibody described herein comprises oneor more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from κ and λ. In some embodiments, a chimeric antibodydescribed herein comprises a human IgG constant region. In someembodiments, a chimeric antibody described herein comprises a human IgG4heavy chain constant region. In some such embodiments, a chimericantibody described herein comprises an S241P mutation in the human IgG4constant region. In some embodiments, a chimeric antibody describedherein comprises a human IgG4 constant region and a human κ light chain.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, a chimericanti-CSF1R antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a chimericanti-CSF1R antibody comprising a human IgG4 or IgG2 heavy chain constantregion is selected.

Exemplary Human Antibodies

Human antibodies can be made by any suitable method. Nonlimitingexemplary methods include making human antibodies in transgenic micethat comprise human immunoglobulin loci. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al., Nature362: 255-8 (1993); Lonberg et al., Nature 368: 856-9 (1994); and U.S.Pat. Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807;6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.

Nonlimiting exemplary methods also include making human antibodies usingphage display libraries. See, e.g., Hoogenboom et al., J. Mol. Biol.227: 381-8 (1992); Marks et al., J. Mol. Biol. 222: 581-97 (1991); andPCT Publication No. WO 99/10494.

In some embodiments, a human anti-CSF1R antibody binds to a polypeptidehaving the sequence of SEQ ID NO: 1. Exemplary human anti-CSF1Rantibodies also include antibodies that compete for binding to CSF1Rwith an antibody described herein. Thus, in some embodiments, a humananti-CSF1R antibody is provided that competes for binding to CSF1R withan antibody selected from Fabs 0301, 0302, and 0311, and bivalent (i.e.,having two heavy chains and two light chains) antibody versions of thoseFabs.

In some embodiments, a human anti-CSF1R antibody comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, the human light chain constant region is of an isotypeselected from κ and λ. In some embodiments, a human antibody describedherein comprises a human IgG constant region. In some embodiments, ahuman antibody described herein comprises a human IgG4 heavy chainconstant region. In some such embodiments, a human antibody describedherein comprises an S241P mutation in the human IgG4 constant region. Insome embodiments, a human antibody described herein comprises a humanIgG4 constant region and a human κ light chain.

In some embodiments, when effector function is desirable, a humananti-CSF1R antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a human anti-CSF1Rantibody comprising a human IgG4 or IgG2 heavy chain constant region isselected.

Additional Exemplary Anti-CSF1R Antibodies

Exemplary anti-CSF1R antibodies also include, but are not limited to,mouse, humanized, human, chimeric, and engineered antibodies thatcomprise, for example, one or more of the CDR sequences describedherein. In some embodiments, an anti-CSF1R antibody comprises a heavychain variable region described herein. In some embodiments, ananti-CSF1R antibody comprises a light chain variable region describedherein. In some embodiments, an anti-CSF1R antibody comprises a heavychain variable region described herein and a light chain variable regiondescribed herein. In some embodiments, an anti-CSF1R antibody comprisesheavy chain CDR1, CDR2, and CDR3 described herein. In some embodiments,an anti-CSF1R antibody comprises light chain CDR1, CDR2, and CDR3described herein. In some embodiments, an anti-CSF1R antibody comprisesheavy chain CDR1, CDR2, and CDR3 described herein and light chain CDR1,CDR2, and CDR3 described herein.

In some embodiments, an anti-CSF1R antibody comprises a heavy chainvariable region of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies also include antibodiescomprising a heavy chain variable region of an antibody selected fromhumanized antibodies huAb1 to huAb16. Nonlimiting exemplary anti-CSF1Rantibodies include antibodies comprising a heavy chain variable regioncomprising a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45.

In some embodiments, an anti-CSF1R antibody comprises a light chainvariable region of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies also include antibodiescomprising a light chain variable region of an antibody selected fromhumanized antibodies huAb1 to huAb16. Nonlimiting exemplary anti-CSF1Rantibodies include antibodies comprising a light chain variable regioncomprising a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to52.

In some embodiments, an anti-CSF1R antibody comprises a heavy chainvariable region and a light chain variable region of an antibodyselected from Fabs 0301, 0302, and 0311. Nonlimiting exemplaryanti-CSF1R antibodies also include antibodies comprising a heavy chainvariable region and a light chain variable region of an antibodyselected from humanized antibodies huAb1 to huAb16. Nonlimitingexemplary anti-CSF1R antibodies include antibodies comprising thefollowing pairs of heavy and light chain variable regions: SEQ ID NOs: 9and 10; SEQ ID NOs: 11 and 12; and SEQ ID NOs: 13 and 14; SEQ ID NOs: 39and 40; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; and SEQ ID NOs: 51 and52. Nonlimiting exemplary anti-CSF1R antibodies also include antibodiescomprising the following pairs of heavy and light chains: SEQ ID NOs: 33and 34; SEQ ID NOs: 35 and 36; and SEQ ID NOs: 37 and 38.

In some embodiments, an anti-CSF1R antibody comprises heavy chain CDR1,CDR2, and CDR3 of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising sets of heavy chain CDR1, CDR2, and CDR3 selected from: SEQID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22, and 23; and SEQ ID NOs: 27,28, and 29.

In some embodiments, an anti-CSF1R antibody comprises light chain CDR1,CDR2, and CDR3 of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising sets of light chain CDR1, CDR2, and CDR3 selected from: SEQID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25, and 26; and SEQ ID NOs: 30,31, and 32.

In some embodiments, an anti-CSF1R antibody comprises heavy chain CDR1,CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 of an antibodyselected from Fabs 0301, 0302, and 0311.

Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising the sets of heavy chain CDR1, CDR2, and CDR3, and light chainCDR1, CDR2, and CDR3 shown above in Table 1.

Further Exemplary Antibodies

In some embodiments, an anti-CSF1R antibody comprises a heavy chaincomprising a variable region sequence that is at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to a sequenceselected from SEQ ID NOs: 9, 11, 13, and 39 to 45, wherein the antibodybinds CSF1R. In some embodiments, an anti-CSF1R antibody comprises alight chain comprising a variable region sequence that is at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52, wherein theantibody binds CSF1R. In some embodiments, an anti-CSF1R antibodycomprises a heavy chain comprising a variable region sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to45; and a light chain comprising a variable region sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to52; wherein the antibody binds CSF1R.

In some embodiments, an anti-CSF1R antibody comprises at least one ofthe CDRs discussed herein. That is, in some embodiments, an anti-CSF1Rantibody comprises at least one CDR selected from a heavy chain CDR1discussed herein, a heavy chain CDR2 discussed herein, a heavy chainCDR3 discussed herein, a light chain CDR1 discussed herein, a lightchain CDR2 discussed herein, and a light chain CDR3 discussed herein.Further, in some embodiments, an anti-CSF1R antibody comprises at leastone mutated CDR based on a CDR discussed herein, wherein the mutated CDRcomprises 1, 2, 3, or 4 amino acid substitutions relative to the CDRdiscussed herein. In some embodiments, one or more of the amino acidsubstitutions are conservative amino acid substitutions. One skilled inthe art can select one or more suitable conservative amino acidsubstitutions for a particular CDR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the antibody comprising the mutated CDR.

Exemplary anti-CSF1R antibodies also include antibodies that compete forbinding to CSF1R with an antibody described herein. Thus, in someembodiments, an anti-CSF1R antibody is provided that competes forbinding to CSF1R with an antibody selected from Fabs 0301, 0302, and0311, and bivalent (i.e., having two heavy chains and two light chains)antibody versions of those Fabs.

Exemplary Antibody Constant Regions

In some embodiments, an antibody described herein comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, the human light chain constant region is of an isotypeselected from κ and λ. In some embodiments, an antibody described hereincomprises a human IgG constant region. In some embodiments, an antibodydescribed herein comprises a human IgG4 heavy chain constant region. Insome such embodiments, an antibody described herein comprises an S241Pmutation in the human IgG4 constant region. In some embodiments, anantibody described herein comprises a human IgG4 constant region and ahuman κ light chain.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, an anti-CSF1Rantibody comprising a human IgG1 heavy chain constant region or a humanIgG3 heavy chain constant region is selected. In some embodiments, wheneffector function is not desirable, an anti-CSF1R antibody comprising ahuman IgG4 or IgG2 heavy chain constant region is selected.

Exemplary Anti-CSF1R Heavy Chain Variable Regions

In some embodiments, anti-CSF1R antibody heavy chain variable regionsare provided. In some embodiments, an anti-CSF1R antibody heavy chainvariable region is a mouse variable region, a human variable region, ora humanized variable region.

An anti-CSF1R antibody heavy chain variable region comprises a heavychain CDR1, FR2, CDR2, FR3, and CDR3. In some embodiments, an anti-CSF1Rantibody heavy chain variable region further comprises a heavy chain FR1and/or FR4. Nonlimiting exemplary heavy chain variable regions include,but are not limited to, heavy chain variable regions having an aminoacid sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45.

In some embodiments, an anti-CSF1R antibody heavy chain variable regioncomprises a CDR1 comprising a sequence selected from SEQ ID NOs: 15, 21,and 27.

In some embodiments, an anti-CSF1R antibody heavy chain variable regioncomprises a CDR2 comprising a sequence selected from SEQ ID NOs: 16, 22,and 28.

In some embodiments, an anti-CSF1R antibody heavy chain variable regioncomprises a CDR3 comprising a sequence selected from SEQ ID NOs: 17, 23,and 29.

Nonlimiting exemplary heavy chain variable regions include, but are notlimited to, heavy chain variable regions comprising sets of CDR1, CDR2,and CDR3 selected from: SEQ ID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22,and 23; and SEQ ID NOs: 27, 28, and 29.

In some embodiments, an anti-CSF1R antibody heavy chain comprises avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 9, 11, 13, and 39 to 45, wherein the heavy chain, togetherwith a light chain, is capable of forming an antibody that binds CSF1R.

In some embodiments, an anti-CSF1R antibody heavy chain comprises atleast one of the CDRs discussed herein. That is, in some embodiments, ananti-CSF1R antibody heavy chain comprises at least one CDR selected froma heavy chain CDR1 discussed herein, a heavy chain CDR2 discussedherein, and a heavy chain CDR3 discussed herein. Further, in someembodiments, an anti-CSF1R antibody heavy chain comprises at least onemutated CDR based on a CDR discussed herein, wherein the mutated CDRcomprises 1, 2, 3, or 4 amino acid substitutions relative to the CDRdiscussed herein. In some embodiments, one or more of the amino acidsubstitutions are conservative amino acid substitutions. One skilled inthe art can select one or more suitable conservative amino acidsubstitutions for a particular CDR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the heavy chain comprising the mutatedCDR.

In some embodiments, a heavy chain comprises a heavy chain constantregion. In some embodiments, a heavy chain comprises a human heavy chainconstant region. In some embodiments, the human heavy chain constantregion is of an isotype selected from IgA, IgG, and IgD. In someembodiments, the human heavy chain constant region is an IgG constantregion. In some embodiments, a heavy chain comprises a human igG4 heavychain constant region. In some such embodiments, the human IgG4 heavychain constant region comprises an S241P mutation.

In some embodiments, when effector function is desirable, a heavy chaincomprises a human IgG1 or IgG3 heavy chain constant region. In someembodiments, when effector function is less desirable, a heavy chaincomprises a human IgG4 or IgG2 heavy chain constant region.

Exemplary Anti-CSF1R Light Chain Variable Regions

In some embodiments, anti-CSF1R antibody light chain variable regionsare provided. In some embodiments, an anti-CSF1R antibody light chainvariable region is a mouse variable region, a human variable region, ora humanized variable region.

An anti-CSF1R antibody light chain variable region comprises a lightchain CDR1, FR2, CDR2, FR3, and CDR3. In some embodiments, an anti-CSF1Rantibody light chain variable region further comprises a light chain FR1and/or FR4. Nonlimiting exemplary light chain variable regions includelight chain variable regions having an amino acid sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52.

In some embodiments, an anti-CSF1R antibody light chain variable regioncomprises a CDR1 comprising a sequence selected from SEQ ID NOs: 18, 24and 30.

In some embodiments, an anti-CSF1R antibody light chain variable regioncomprises a CDR2 comprising a sequence selected from SEQ ID NOs: 19, 25,and 31.

In some embodiments, an anti-CSF1R antibody light chain variable regioncomprises a CDR3 comprising a sequence selected from SEQ ID NOs: 20, 26,and 32.

Nonlimiting exemplary light chain variable regions include, but are notlimited to, light chain variable regions comprising sets of CDR1, CDR2,and CDR3 selected from: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25,and 26; and SEQ ID NOs: 30, 31, and 32.

In some embodiments, an anti-CSF1R antibody light chain comprises avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52, wherein the light chain, togetherwith a heavy chain, is capable of forming an antibody that binds CSF1R.

In some embodiments, an anti-CSF1R antibody light chain comprises atleast one of the CDRs discussed herein. That is, in some embodiments, ananti-CSF1R antibody light chain comprises at least one CDR selected froma light chain CDR1 discussed herein, a light chain CDR2 discussedherein, and a light chain CDR3 discussed herein. Further, in someembodiments, an anti-CSF1R antibody light chain comprises at least onemutated CDR based on a CDR discussed herein, wherein the mutated CDRcomprises 1, 2, 3, or 4 amino acid substitutions relative to the CDRdiscussed herein. In some embodiments, one or more of the amino acidsubstitutions are conservative amino acid substitutions. One skilled inthe art can select one or more suitable conservative amino acidsubstitutions for a particular CDR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the light chain comprising the mutatedCDR.

In some embodiments, a light chain comprises a human light chainconstant region. In some embodiments, a human light chain constantregion is selected from a human κ and a human λ, light chain constantregion.

Exemplary Additional CSF1R Binding Molecules

In some embodiments, additional molecules that bind CSF1R are provided.Such molecules include, but are not limited to, non-canonical scaffolds,such as anti-calins, adnectins, ankyrin repeats, etc. See, e.g., Hosseet al., Prot. Sci. 15:14 (2006); Fiedler, M. and Skerra, A.,“Non-Antibody Scaffolds,” pp. 467-499 in Handbook of TherapeuticAntibodies, Dubel, S., ed., Wiley-VCH, Weinheim, Germany, 2007.

Exemplary Properties of Anti-CSF1R Antibodies

In some embodiments, an antibody having a structure described abovebinds to the CSF1R with a binding affinity (K_(D)) of less than 1 nM,blocks binding of CSF1 and/or IL-34 to CSF1R, and inhibits CSF1Rphosphorylation induced by CSF1 and/or IL-34.

In some embodiments, an anti-CSF1R antibody binds to the extracellulardomain of CSF1R (CSF1R-ECD). In some embodiments, an anti-CSF1R antibodyhas a binding affinity (K_(D)) for CSF1R of less than 1 nM, less than0.5 nM, less than 0.1 nM, or less than 0.05 nM. In some embodiments, ananti-CSF1R antibody has a K_(D) of between 0.01 and 1 nM, between 0.01and 0.5 nM, between 0.01 and 0.1 nM, between 0.01 and 0.05 nM, orbetween 0.02 and 0.05 nM.

In some embodiments, an anti-CSF1R antibody blocks ligand binding toCSF1R. In some embodiments, an anti-CSF1R antibody blocks binding ofCSF1 to CSF1R. In some embodiments, an anti-CSF1R antibody blocksbinding of IL-34 to CSF1R. In some embodiments, an anti-CSF1R antibodyblocks binding of both CSF1 and IL-34 to CSF1R. In some embodiments, anantibody that blocks ligand binding binds to the extracellular domain ofCSF1R. In some embodiments, an antibody blocks ligand binding to CSF1Rwhen it reduces the amount of detectable binding of a ligand to CSF1R byat least 50%, using the assay described, e.g., U.S. Pat. No. 8,206,715B2, Example 7, which is incorporated herein by reference for anypurpose. In some embodiments, an antibody reduces the amount ofdetectable binding of a ligand to CSF1R by at least 60%, at least 70%,at least 80%, or at least 90%. In some such embodiments, the antibody issaid to block ligand binding by at least 50%, at least 60%, at least70%, etc.

In some embodiments, an anti-CSF1R antibody inhibits ligand-inducedCSF1R phosphorylation. In some embodiments, an anti-CSF1R antibodyinhibits CSF1-induced CSF1R phosphorylation. In some embodiments, ananti-CSF1R antibody inhibits IL-34-induced CSF1R phosphorylation. Insome embodiments, an anti-CSF1R antibody inhibits both CSF1-induced andIL-34-induced CSF1R phosphorylation. In some embodiments, an antibody isconsidered to “inhibit ligand-induced CSF1R phosphorylation” when itreduces the amount of detectable ligand-induced CSF1R phosphorylation byat least 50%, using the assay described, e.g., U.S. Pat. No. 8,206,715B2, Example 6, which is incorporated herein by reference for anypurpose. In some embodiments, an antibody reduces the amount ofdetectable ligand-induced CSF1R phosphorylation by at least 60%, atleast 70%, at least 80%, or at least 90%. In some such embodiments, theantibody is said to inhibit ligand-induced CSF1R phosphorylation by atleast at least 50%, at least 60%, at least 70%, etc.

In some embodiments, an antibody inhibits monocyte proliferation and/orsurvival responses in the presence of CSF1 and/or IL-34. In someembodiments, an antibody is considered to “inhibit monocyteproliferation and/or survival responses” when it reduces the amount ofmonocyte proliferation and/or survival responses in the presence of CSF1and/or IL-34 by at least 50%, using the assay described, e.g., U.S. Pat.No. 8,206,715 B2, Example 10, which is incorporated herein by referencefor any purpose. In some embodiments, an antibody reduces the amount ofmonocyte proliferation and/or survival responses in the presence of CSF1and/or IL-34 by at least 60%, at least 70%, at least 80%, or at least90%. In some such embodiments, the antibody is said to inhibit monocyteproliferation and/or survival responses by at least at least 50%, atleast 60%, at least 70%, etc.

Exemplary Immune Stimulating Agents

Immune stimulating agents may include, for example, a small moleculedrug, antibody or fragment thereof, or other biologic or small molecule.Examples of biologic immune stimulating agents include, but are notlimited to, antibodies, antibody fragments, fragments of receptor orligand polypeptides, for example that block receptor-ligand binding,vaccines and cytokines. In one aspect, the antibody is a monoclonalantibody. In certain aspects, the monoclonal antibody is humanized orhuman antibody.

In some embodiments, the at least one immune stimulating agent comprisesan agonist of an immune stimulatory molecule, including a co-stimulatorymolecule, while in some embodiments, the at least one immune stimulatingagent comprises an antagonist of an immune inhibitory molecule,including a co-inhibitory molecule. In some embodiments, the at leastone immune stimulating agent comprises an agonist of animmune-stimulatory molecule, including a co-stimulatory molecule, foundon immune cells, such as T cells. In some embodiments, the at least oneimmune stimulating agent comprises an antagonist of an immune inhibitorymolecule, including a co-inhibitory molecule, found on immune cells,such as T cells. In some embodiments, the at least one immunestimulating agent comprises an agonist of an immune stimulatorymolecule, including a co-stimulatory molecule, found on cells involvedin innate immunity, such as NK cells. In some embodiments, the at leastone immune stimulating agent comprises an antagonist of an immuneinhibitory molecule, including a co-inhibitory molecule, found on cellsinvolved in innate immunity, such as NK cells. In some embodiments, thecombination enhances the antigen-specific T cell response in the treatedsubject and/or enhances the innate immunity response in the subject. Insome embodiments, the combination results in an improved anti-tumorresponse in an animal cancer model, such as a xenograft model, comparedto administration of either the anti-CSF1R antibody or immunestimulating agent alone. In some embodiments, the combination results ina synergistic response in an animal cancer model, such as a xenograftmodel, compared to administration of either the anti-CSF1R antibody orimmune stimulating agent alone.

In certain embodiments, an immune stimulating agent targets astimulatory or inhibitory molecule that is a member of theimmunoglobulin super family (IgSF). For example, an immune stimulatingagent may be an agent that targets (or binds specifically to) a memberof the B7 family of membrane-bound ligands, which includes B7-1, B7-2,B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6, or aco-stimulatory or co-inhibitory receptor binding specifically to a B7family member. An immune stimulating agent may be an agent that targetsa member of the TNF family of membrane bound ligands or a co-stimulatoryor co-inhibitory receptor binding specifically to a member of the TNFfamily. Exemplary TNF and TNFR family members that may be targeted byimmune stimulating agents include CD40 and CD40L, OX-40, OX-40L, GITR,GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L,TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL,TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT,DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1,Lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, Lymphotoxin α 1β2, FAS, FASL,RELT, DR6, TROY and NGFR. An immune stimulating agent may be an agent,e.g., an antibody, targeting an IgSF member, such as a B7 family member,a B7 receptor family member, a TNF family member or a TNFR familymember, such as those described above.

In some embodiments, an immune stimulating agent may comprise (i) anantagonist of a protein that inhibits T cell activation (e.g., immunecheckpoint inhibitor) such as CTLA-4, LAG-3, TIM3, Galectin 9, CEACAM-1,BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4,CD48, GARP, PD1H, LAIR1, TIM-1, TIM-4, and ILT4 and/or may comprise (ii)an agonist of a protein that stimulates T cell activation such as B7-1,B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR,GITRL, CD70, CD27, CD40, CD40L, DR3 and CD28H.

In some embodiments, an immune stimulating agent may comprise an agentthat inhibit or is an antagonist of a cytokine that inhibits T cellactivation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressivecytokines), and it some embodiments an immune stimulating agent maycomprise an agent that is an agonist of a cytokine, such as IL-2, IL-7,IL-12, IL-15, IL-21 and IFNα (e.g., the cytokine itself) that stimulatesT cell activation. In some embodiments, immune stimulating agents maycomprise an antagonist of a chemokine, such as CXCR2 (e.g., MK-7123),CXCR4 (e.g. AMD3100), CCR2, or CCR4 (mogamulizumab).

In some embodiments, immune stimulating agents may include antagonistsof inhibitory receptors on NK cells or agonists of activating receptorson NK cells. For example, an anti-CSF1R antibody can be combined with anantagonist of KIR, optionally along with at least one other immunestimulating agent such as an agonist of CD40.

Immune stimulating agents may also include agents that inhibit TGF-βsignaling, agents that enhance tumor antigen presentation, e.g.,dendritic cell vaccines, GM-CSF secreting cellular vaccines, CpGoligonucleotides, and imiquimod, or therapies that enhance theimmunogenicity of tumor cells (e.g., anthracyclines).

Immune stimulating agents may also include certain vaccines such asmesothelin-targeting vaccines or attenuated listeria cancer vaccines,such as CRS-207.

Immune stimulating agents may also comprise agents that deplete or blockTreg cells, such as agents that specifically bind to CD25.

Immune stimulating agents may also comprise agents that inhibit ametabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase,arginase, or nitric oxide synthetase.

Immune stimulating agents may also comprise agents that inhibit theformation of adenosine or inhibit the adenosine A2A receptor.

Immune stimulating agents may also comprise agents that reverse/preventT cell anergy or exhaustion and agents that trigger an innate immuneactivation and/or inflammation at a tumor site.

An anti-CSF1R antibody may be combined with more than one immunestimulating agent, such as a CD40 agonist and at least one additionalimmune stimulating agent. The anti-CSF1R antibody, optionally along witha CD40 agonist, may be combined with a combinatorial approach thattargets multiple elements of the immune pathway, such as one or more ofthe following: at least one agent that enhances tumor antigenpresentation (e.g., dendritic cell vaccine, GM-CSF secreting cellularvaccines, CpG oligonucleotides, imiquimod); at least one agent thatinhibits negative immune regulation e.g., by inhibiting CTLA-4 pathwayand/or depleting or blocking Treg or other immune suppressing cells; atherapy that stimulates positive immune regulation, e.g., with agoniststhat stimulate the CD-137, OX-40 and/or GITR pathway and/or stimulate Tcell effector function; at least one agent that increases systemicallythe frequency of anti-tumor T cells; a therapy that depletes or inhibitsTregs, such as Tregs in the tumor, e.g., using an antagonist of CD25(e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; at least oneagent that impacts the function of suppressor myeloid cells in thetumor; a therapy that enhances immunogenicity of tumor cells (e.g.,anthracyclines); adoptive T cell or NK cell transfer includinggenetically modified cells, e.g., cells modified by chimeric antigenreceptors (CAR-T therapy); at least one agent that inhibits a metabolicenzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase ornitric oxide synthetase; at least one agent that reverses/prevents Tcell anergy or exhaustion; a therapy that triggers an innate immuneactivation and/or inflammation at a tumor site; administration of immunestimulatory cytokines or blocking of immuno repressive cytokines.

For example, an anti-CSF1R antibody, optionally with a CD40 agonist, canbe used with one or more agonistic agents that ligate positivecostimulatory receptors; one or more antagonists (blocking agents) thatattenuate signaling through inhibitory receptors, such as antagoniststhat overcome distinct immune suppressive pathways within the tumormicroenvironment; one or more agents that increase systemically thefrequency of anti-tumor immune cells, such as T cells, deplete orinhibit Tregs (e.g., by inhibiting CD25); one or more agents thatinhibit metabolic enzymes such as IDO; one or more agents thatreverse/prevent T cell anergy or exhaustion; and one or more agents thattrigger innate immune activation and/or inflammation at tumor sites.

In one embodiment, the at least one immune stimulating agent comprises aCTLA-4 antagonist, such as an antagonistic CTLA-4 antibody. SuitableCTLA-4 antibodies include, for example, YERVOY (ipilimumab) ortremelimumab.

In some embodiments, the at least one immune stimulating agent comprisesa LAG-3 antagonist, such as an antagonistic LAG-3 antibody. SuitableLAG3 antibodies include, for example, BMS-986016 (WO10/19570,WO14/08218), or IMP-731 or IMP-321 (WO08/132601, WO09/44273).

In some embodiments, the at least one immune stimulating agent comprisesa CD137 (4-1BB) agonist, such as an agonistic CD137 antibody. SuitableCD137 antibodies include, for example, urelumab or PF-05082566(WO12/32433).

In some embodiments, the at least one immune stimulating agent comprisesa GITR agonist, such as an agonistic GITR antibody. Suitable GITRantibodies include, for example, TRX-518 (WO06/105021, WO09/009116),MK-4166 (WO11/028683) or a GITR antibody disclosed in WO2015/031667.

In some embodiments, the at least one immune stimulating agent comprisesan OX40 agonist, such as an agonistic OX40 antibody. Suitable OX40antibodies include, for example, MEDI-6383, MEDI-6469 or MOXR0916(RG7888; WO06/029879).

In some embodiments, the at least one immune stimulating agent comprisesa CD27 agonist, such as an agonistic CD27 antibody. Suitable CD27antibodies include, for example, varlilumab (CDX-1127).

In some embodiments, the at least one immune stimulating agent comprisesMGA271, which targets B7H3 (WO11/109400).

In some embodiments, the at least one immune stimulating agent comprisesa KIR antagonist, such as lirilumab.

In some embodiments, the at least one immune stimulating agent comprisesan IDO antagonist. IDO antagonists include, for example, INCB-024360(WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919(WO09/73620, WO09/1156652, WO11/56652, WO12/142237) or F001287.

In some embodiments, the at least one immune stimulating agent comprisesa Toll-like receptor agonist, e.g., a TLR2/4 agonist (e.g., BacillusCalmette-Guerin); a TLR7 agonist (e.g., Hiltonol or Imiquimod); a TLR7/8agonist (e.g., Resiquimod); or a TLR9 agonist (e.g., CpG7909).

In some embodiments, the at least one immune stimulating agent comprisesa TGF-β inhibitor, e.g., GC1008, LY2157299, TEW7197 or IMC-TR1.

CD40 Agonists and Exemplary CD40 Agonist Molecules

In some embodiments, the at least one immune stimulating agent comprisesa CD40 agonist, optionally together with at least one additional immunestimulating agent as described herein. The cell surface molecule CD40 isa member of the tumor necrosis factor receptor superfamily and isexpressed by antigen presenting cells such as dentritic cells, B cells,macrophages and monocytes and is also expressed on other cell types,including immune, hematopoietic, vascular, and epithelial cells, as wellas on various tumor cells. In antigen presenting cells CD40 signalingresults in activation and upregulation of T cell costimulatory moleculesand other critical immune mediators required for the induction of animmune response. Agonists of CD40 are potential cancer therapies,causing tumor regression through both anti-tumor immune activation anddirect cytotoxic effect on tumor cells. CD40-targeting therapies haveundergone phase 1 clinical evaluation in advanced-stage cancer patients,and initial findings have shown efficacy in the absence of majortoxicity.

With regard to CD40, for example, animal models have shown that ligationof CD40 on dendritic cells results in activation of cytotoxic Tlymphocytes that mediate tumor killing (Marzo et al., 2000, J. Immunol.;Todryk et al., 2001, J. Immunol. Methods.) Activation of CD40 onmacrophages results in tumoralcidal activity (Beatty et al., 2011,Science), and cytokines produced from CD40 stimulated antigen presentingcells leads to the activation of natural killer cells important fortumor eradication. Given the complex nature of an anti-tumor immuneresponse, effective cancer therapy may require combining multipleimmunotherapy agents. Consistent with this, small molecule inhibition ofCSF1R was shown to synergize with anti-PD1 immune checkpoint blockade ina pancreatic tumor model. See Zhu et al., 2014, Cancer Res., 74:5057-5069. Thus, tumors that have CSF1R-expressing TAMs may be sensitiveto combination therapy with an anti-CSF1R antibody and a CD40 agonist.

Exemplary CD40 agonists of the compositions and methods of thisinvention include, for example, anti-CD40 antibodies that enhance CD40activity. Such antibodies may be humanized antibodies, chimericantibodies, mouse antibodies, human antibodies, and antibodiescomprising the heavy chain and/or light chain CDRs of an anti-CD40antibody discussed herein.

Various agonist anti-CD40 antibodies are known in the art. Nonlimitingexemplary agonist anti-CD40 antibodies include, but are not limited to,CP-870,893 (Pfizer and VLST; antibody 21.4.1 in EP 1 476 185 B1 and U.S.Pat. No. 7,338,660; see also clinicaltrials.gov/ct2/show/NCT02225002);dacetuzumab (Seattle Genetics; SEQ ID NOs: 98 and 99 herein; see alsoU.S. Pat. Nos. 6,946,129 and 8,303,955); RO7009789 (Roche; see, e.g.,clinicaltrials.gov/ct2/show/NCT02304393); ADC-1013 (AlligatorBioscience; US Publication No. 2014/0348836; see alsoclinicaltrials.gov/ct2/show/NCT02379741); SEA-CD40 (Seattle Genetics;afucosylated form of antibody comprising SEQ ID NOs: 98 and 99; see alsoclinicaltrials.gov/ct2/show/NCT02376699); and Chi Lob 7/4 (Univ.Southampton; US Publication No. 2009/0074711; see alsoclinicaltrials.gov/ct2/show/NCT01561911). See, e.g., Vonderheide et al.,2013, Clin Cancer Res 19:1035.

Exemplary CD40 agonists also include recombinant CD40L.

Exemplary Antibody Conjugates

In some embodiments, an antibody is conjugated to a label and/or acytotoxic agent. As used herein, a label is a moiety that facilitatesdetection of the antibody and/or facilitates detection of a molecule towhich the antibody binds. Nonlimiting exemplary labels include, but arenot limited to, radioisotopes, fluorescent groups, enzymatic groups,chemiluminescent groups, biotin, epitope tags, metal-binding tags, etc.One skilled in the art can select a suitable label according to theintended application.

As used herein, a cytotoxic agent is a moiety that reduces theproliferative capacity of one or more cells. A cell has reducedproliferative capacity when the cell becomes less able to proliferate,for example, because the cell undergoes apoptosis or otherwise dies, thecell fails to proceed through the cell cycle and/or fails to divide, thecell differentiates, etc. Nonlimiting exemplary cytotoxic agentsinclude, but are not limited to, radioisotopes, toxins, andchemotherapeutic agents. One skilled in the art can select a suitablecytotoxic according to the intended application.

In some embodiments, a label and/or a cytotoxic agent is conjugated toan antibody using chemical methods in vitro. Nonlimiting exemplarychemical methods of conjugation are known in the art, and includeservices, methods and/or reagents commercially available from, e.g.,Thermo Scientific Life Science Research Produces (formerly Pierce;Rockford, Ill.), Prozyme (Hayward, Calif.), SACRI Antibody Services(Calgary, Canada), AbD Serotec (Raleigh, N.C.), etc. In someembodiments, when a label and/or cytotoxic agent is a polypeptide, thelabel and/or cytotoxic agent can be expressed from the same expressionvector with at least one antibody chain to produce a polypeptidecomprising the label and/or cytotoxic agent fused to an antibody chain.One skilled in the art can select a suitable method for conjugating alabel and/or cytotoxic agent to an antibody according to the intendedapplication.

Exemplary Leader Sequences

In order for some secreted proteins to express and secrete in largequantities, a leader sequence from a heterologous protein may bedesirable. In some embodiments, a leader sequence is selected from SEQID NOs: 3 and 4, which are light chain and heavy chain leader sequences,respectively. In some embodiments, employing heterologous leadersequences may be advantageous in that a resulting mature polypeptide mayremain unaltered as the leader sequence is removed in the ER during thesecretion process. The addition of a heterologous leader sequence may berequired to express and secrete some proteins.

Certain exemplary leader sequence sequences are described, e.g., in theonline Leader sequence Database maintained by the Department ofBiochemistry, National University of Singapore. See Choo et al., BMCBioinformatics, 6: 249 (2005); and PCT Publication No. WO 2006/081430.

Nucleic Acid Molecules Encoding Antibodies

Nucleic acid molecules comprising polynucleotides that encode one ormore chains of an antibody are provided. In some embodiments, a nucleicacid molecule comprises a polynucleotide that encodes a heavy chain or alight chain of an antibody. In some embodiments, a nucleic acid moleculecomprises both a polynucleotide that encodes a heavy chain and apolynucleotide that encodes a light chain, of an antibody. In someembodiments, a first nucleic acid molecule comprises a firstpolynucleotide that encodes a heavy chain and a second nucleic acidmolecule comprises a second polynucleotide that encodes a light chain.

In some such embodiments, the heavy chain and the light chain areexpressed from one nucleic acid molecule, or from two separate nucleicacid molecules, as two separate polypeptides. In some embodiments, suchas when an antibody is an scFv, a single polynucleotide encodes a singlepolypeptide comprising both a heavy chain and a light chain linkedtogether.

In some embodiments, a polynucleotide encoding a heavy chain or lightchain of an antibody comprises a nucleotide sequence that encodes aleader sequence, which, when translated, is located at the N terminus ofthe heavy chain or light chain. As discussed above, the leader sequencemay be the native heavy or light chain leader sequence, or may beanother heterologous leader sequence.

Nucleic acid molecules may be constructed using recombinant DNAtechniques conventional in the art. In some embodiments, a nucleic acidmolecule is an expression vector that is suitable for expression in aselected host cell.

Antibody Expression and Production

Vectors

Vectors comprising polynucleotides that encode antibody heavy chainsand/or light chains are provided. Vectors comprising polynucleotidesthat encode antibody heavy chains and/or light chains are also provided.Such vectors include, but are not limited to, DNA vectors, phagevectors, viral vectors, retroviral vectors, etc. In some embodiments, avector comprises a first polynucleotide sequence encoding a heavy chainand a second polynucleotide sequence encoding a light chain. In someembodiments, the heavy chain and light chain are expressed from thevector as two separate polypeptides. In some embodiments, the heavychain and light chain are expressed as part of a single polypeptide,such as, for example, when the antibody is an scFv.

In some embodiments, a first vector comprises a polynucleotide thatencodes a heavy chain and a second vector comprises a polynucleotidethat encodes a light chain. In some embodiments, the first vector andsecond vector are transfected into host cells in similar amounts (suchas similar molar amounts or similar mass amounts). In some embodiments,a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and thesecond vector is transfected into host cells. In some embodiments, amass ratio of between 1:1 and 1:5 for the vector encoding the heavychain and the vector encoding the light chain is used. In someembodiments, a mass ratio of 1:2 for the vector encoding the heavy chainand the vector encoding the light chain is used.

In some embodiments, a vector is selected that is optimized forexpression of polypeptides in CHO or CHO-derived cells, or in NSO cells.Exemplary such vectors are described, e.g., in Running Deer et al.,Biotechnol. Prog. 20:880-889 (2004).

In some embodiments, a vector is chosen for in vivo expression ofantibody heavy chains and/or antibody light chains in animals, includinghumans. In some such embodiments, expression of the polypeptide is underthe control of a promoter that functions in a tissue-specific manner.For example, liver-specific promoters are described, e.g., in PCTPublication No. WO 2006/076288.

Host Cells

In various embodiments, antibody heavy chains and/or light chains may beexpressed in prokaryotic cells, such as bacterial cells; or ineukaryotic cells, such as fungal cells (such as yeast), plant cells,insect cells, and mammalian cells. Such expression may be carried out,for example, according to procedures known in the art. Exemplaryeukaryotic cells that may be used to express polypeptides include, butare not limited to, COS cells, including COS 7 cells; 293 cells,including 293-6E cells; CHO cells, including CHO—S and DG44 cells;PER.C6® cells (Crucell); and NSO cells. In some embodiments, antibodyheavy chains and/or light chains may be expressed in yeast. See, e.g.,U.S. Publication No. US 2006/0270045 A1. In some embodiments, aparticular eukaryotic host cell is selected based on its ability to makedesired post-translational modifications to the antibody heavy chainsand/or light chains. For example, in some embodiments, CHO cells producepolypeptides that have a higher level of sialylation than the samepolypeptide produced in 293 cells.

Introduction of one or more nucleic acids into a desired host cell maybe accomplished by any method, including but not limited to, calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,etc. Nonlimiting exemplary methods are described, e.g., in Sambrook etal., Molecular Cloning, A Laboratory Manual, 3^(rd) ed. Cold SpringHarbor Laboratory Press (2001). Nucleic acids may be transiently orstably transfected in the desired host cells, according to any suitablemethod.

In some embodiments, one or more polypeptides may be produced in vivo inan animal that has been engineered or transfected with one or morenucleic acid molecules encoding the polypeptides, according to anysuitable method.

Purification of Antibodies

Antibodies may be purified by any suitable method. Such methods include,but are not limited to, the use of affinity matrices or hydrophobicinteraction chromatography. Suitable affinity ligands include theantigen and ligands that bind antibody constant regions. For example, aProtein A, Protein G, Protein A/G, or an antibody affinity column may beused to bind the constant region and to purify an antibody. Hydrophobicinteractive chromatography, for example, a butyl or phenyl column, mayalso suitable for purifying some polypeptides. Many methods of purifyingpolypeptides are known in the art.

Cell-Free Production of Antibodies

In some embodiments, an antibody is produced in a cell-free system.Nonlimiting exemplary cell-free systems are described, e.g., inSitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, TrendsBiotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713(2003).

Therapeutic Compositions and Methods

Methods of Treating Cancer

In some embodiments, methods for treating cancer are provided,comprising administering an effective amount of an anti-CSF1R antibodyand an effective amount of at least one immune stimulating agent. Insome embodiments, the anti-CSF1R antibody and the at least one immunestimulating agent are administered concurrently. For example, thetherapeutics may be infused together or injected at roughly the sametime. In some embodiments, the anti-CSF1R antibody and the at least oneimmune stimulating agent administered sequentially. For example, in someembodiments the anti-CSF1R antibody is administered sequentially beforeor after at least one immune stimulating agent such that the twotherapeutics are administered 30 minutes, 60 minutes, 90 minutes, 120minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3days, 5 days, 7 days, or two weeks apart.

In some embodiments, at least one, at least two, at least three doses,at least five doses, or at least ten doses of an anti-CSF1R antibody isadministered prior to administration of at least one immune stimulatingagent. In some embodiments, at least one, at least two, at least threedoses, at least five doses, or at least ten doses of at least one immunestimulating agent is administered prior to administration of ananti-CSF1R antibody. In some embodiments, the last dose of immunestimulating agent is administered at least one, two, three, five, daysor ten, or one, two, three, five, twelve, or twenty four weeks prior tothe first dose of CSFR1 inhibitor. In some other embodiment, the lastdose of CSFR1 inhibitor is administered at least one, two, three, five,days or ten, or one, two, three, five, twelve, or twenty four weeksprior to the first dose of at least one immune stimulating agent. Insome embodiments, a subject has received, or is receiving, therapy withat least one immune stimulating agent, and an anti-CSF1R antibody isadded to the therapeutic regimen.

In some embodiments, a method of selecting a patient for combinationtherapy with an anti-CSF1R antibody and a at least one immunestimulating agent, such as a CD40 agonist is provided, comprisingdetermining the levels of TAMs and/or CD8+ T cells in the patient. Insome embodiments, if a patient's TAM levels are high, the patient isselected for combination therapy. In some embodiments, if a patient'sTAM and CD8+ T cell levels are high, the patient is selected forcombination therapy. The level of TAMs or CD8+ T cells is considered“high” if it is at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 75%, or at least 100% higher than the level in anindividual who does not have cancer. In some embodiments, the level ofTAMs or CD8+ T cells is considered “high” if it is above the medianlevel found in individuals with cancer. In some embodiments, if apatient's TAM levels are high and CD8+ T cell levels are low, thepatient is selected for combination therapy with an anti-CSF1R antibodyand at least one immune stimulating agent, such as a CD40 agonist. Thelevel of CD8+ T cells is considered “low” if it is at or below themedian level found in individuals with cancer. In some embodiment, thelevel of CD8+ T cells is considered “low” if it is at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 75%, or atleast 100% lower than the level in an individual who does not havecancer. In some embodiments, expression of CSF1R on the patient's TAMsis determined. In some embodiments, if the patient's TAMs express CSF1R,the patient is selected for combination therapy. In some embodiments, ifthe patient's TAMs express elevated levels of CSF1R, the patient isselected for combination therapy. In some embodiments, a patient's TAMsare considered to express “elevated” levels of CSF1R if the level ofCSF1R is at or above the median level of CSF1R found expressed on TAMSin individuals with cancer. In some embodiments, if the patient's CSF1Rexpression shows a high correlation with the level of CD8+ T cells, thepatient is selected for combination therapy. The correlation of theexpressions is considered “high” if it is at or above the median levelfound in individuals with cancer.

Levels of TAMs, CSF1R expression, CD8+ T cells, and/or regulatory Tcells may be measured by methods in the art. Nonexemplary methodsinclude immunohistochemistry (IHC), fluorescence-activated cell sorting(FACS), protein arrays, and gene expression assays, such as RNAsequencing, gene arrays, and quantitative PCR. In some embodiments, oneor more markers selected from CSF1R, CD68, CD163, CD8, and FoxP3 may bedetected by IHC, FACS, or gene expression assay on tumor sections, ordissociated cells from tumor sections.

In some embodiments, the cancer is selected from squamous cell cancer,small-cell lung cancer, pituitary cancer, esophageal cancer,astrocytoma, soft tissue sarcoma, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, renal cancer, liver cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma, brain cancer,endometrial cancer, testis cancer, cholangiocarcinoma, gallbladdercarcinoma, gastric cancer, melanoma, and various types of head and neckcancer. In some embodiments, lung cancer is non-small cell lung canceror lung squamous cell carcinoma. In some embodiments, leukemia is acutemyeloid leukemia or chronic lymphocytic leukemia. In some embodiments,breast cancer is breast invasive carcinoma. In some embodiments, ovariancancer is ovarian serous cystadenocarcinoma. In some embodiments, kidneycancer is kidney renal clear cell carcinoma. In some embodiments, coloncancer is colon adenocarcinoma. In some embodiments, bladder cancer isbladder urothelial carcinoma. In some embodiments, the cancer isselected from bladder cancer, cervical cancer (such as squamous cellcervical cancer), head and neck squamous cell carcinoma, rectaladenocarcinoma, non-small cell lung cancer, endometrial cancer, prostateadenocarcinoma, colon cancer, ovarian cancer (such as serous epithelialovarian cancer), and melanoma.

In some embodiments, the anti-CSF1R antibody blocks binding of CSF1and/or IL-34 to CSF1R and/or inhibits CSF1R phosphorylation induced byCSF1 and/or IL-34. In some embodiments, the anti-CSF1R antibody locksbinding of CSF1 and IL-34 to CSF1R and/or inhibits CSF1R phosphorylationinduced by CSF1 and/or IL-34. In some embodiments, the anti-CSF1Rantibody comprises the CDRs of, or the variable regions of, an antibodyselected from huAb1 to huAb16, described herein. In some embodiments,the anti-CSF1R antibody comprises the CDRs of, or the variable regionsof, huAb1.

In some embodiments, the at least one immune stimulating agent comprisesan antagonist of an inhibitor of the activation of T cells, while insome embodiments, the at least one immune stimulating agent comprisescomprises an agonist of a stimulator of the activation of T cells. Insome embodiments, the at least one immune stimulating agent comprises anantagonist of CTLA4, LAG-3, Galectin 1, Galectin 9, CEACAM-1, BTLA,CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP,PD1H, LAIR1, TIM1, TIM3, TIM4, ILT4, IL-6, IL-10, TGFβ, VEGF, KIR,LAG-3, adenosine A2A receptor, PI3Kdelta, or IDO. In some embodiments,the at least one immune stimulating agent comprises an agonist of B7-1,B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR,GITRL, CD27, CD40, CD40L, DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21,IFNα, STING, or a Toll-like receptor agonist such as a TLR2/4 agonist.In some embodiments, the at least one immune stimulating agent comprisesan agent that binds to a member of the B7 family of membrane-boundproteins such as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5(VISTA), and B7-H6. In some embodiments, the at least one immunestimulating agent comprises an agent that binds to a member of the TNFreceptor family or a co-stimulatory or co-inhibitory molecule binding toa member of the TNF receptor family such as CD40, CD40L, OX40, OX40L,GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB),TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK,RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, EDA1, EDA2, TACI, APRIL,BCMA, LTβR, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFβ, TNFR2,TNFα, 1β2, FAS, FASL, RELT, DR6, TROY, or NGFβ. In some embodiments, theat least one immune stimulating agent comprises an agent thatantagonizes or inhibits a cytokine that inhibits T cell activation suchas IL-6, IL-10, TGFβ, VEGF. In some embodiments, the at least one immunestimulating agent comprises an agonist of a cytokine that stimulates Tcell activation such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα. Insome embodiments, the at least one immune stimulating agent comprises anantagonist of a chemokine, such as CXCR2, CXCR4, CCR2, or CCR4. In someembodiments, the at least one immune stimulating agent comprises anantibody. In some embodiments, the at least one immune stimulating agentmay comprise a vaccine, such as a mesothelin-targeting vaccine orattenuated listeria cancer vaccine such as CRS-207.

In some embodiments, the at least one immune stimulating agent comprisesa CD40 agonist, for example, an anti-CD40 antibody. Nonlimitingexemplary agonist anti-CD40 antibodies include CP-870,893 (Pfizer andVLST); dacetuzumab (Seattle Genetics); RO7009789 (Roche); ACD-1013(Alligator Bioscience); SEA-CD40 (Seattle Genetics); and Chi Lob 7/4(Univ. Southampton). In some embodiments, a CD40 agonist is recombinantCD40L.

Routes of Administration and Carriers

In various embodiments, antibodies may be administered in vivo byvarious routes, including, but not limited to, oral, intra-arterial,parenteral, intranasal, intramuscular, intracardiac, intraventricular,intratracheal, buccal, rectal, intraperitoneal, intradermal, topical,transdermal, and intrathecal, or otherwise by implantation orinhalation. The subject compositions may be formulated into preparationsin solid, semi-solid, liquid, or gaseous forms; including, but notlimited to, tablets, capsules, powders, granules, ointments, solutions,suppositories, enemas, injections, inhalants, and aerosols. A nucleicacid molecule encoding an antibody may be coated onto goldmicroparticles and delivered intradermally by a particle bombardmentdevice, or “gene gun,” as described in the literature (see, e.g., Tanget al., Nature 356:152-154 (1992)). The appropriate formulation androute of administration may be selected according to the intendedapplication.

In various embodiments, compositions comprising antibodies are providedin formulations with a wide variety of pharmaceutically acceptablecarriers (see, e.g., Gennaro, Remington: The Science and Practice ofPharmacy with Facts and Comparisons: Drugfacts Plus, 20^(th) ed. (2003);Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems,7^(th) ed., Lippencott Williams and Wilkins (2004); Kibbe et al.,Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press(2000)). Various pharmaceutically acceptable carriers, which includevehicles, adjuvants, and diluents, are available. Moreover, variouspharmaceutically acceptable auxiliary substances, such as Ph adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are also available. Non-limiting exemplary carriersinclude saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof.

In various embodiments, compositions comprising antibodies may beformulated for injection, including subcutaneous administration, bydissolving, suspending, or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other oils, synthetic aliphatic acidglycerides, esters of higher aliphatic acids, or propylene glycol; andif desired, with conventional additives such as solubilizers, isotonicagents, suspending agents, emulsifying agents, stabilizers andpreservatives. In various embodiments, the compositions may beformulated for inhalation, for example, using pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen, and thelike. The compositions may also be formulated, in various embodiments,into sustained release microcapsules, such as with biodegradable ornon-biodegradable polymers. A non-limiting exemplary biodegradableformulation includes poly lactic acid-glycolic acid polymer. Anon-limiting exemplary non-biodegradable formulation includes apolyglycerin fatty acid ester. Certain methods of making suchformulations are described, for example, in EP 1 125 584 A1.

Pharmaceutical packs and kits comprising one or more containers, eachcontaining one or more doses of an antibody or combination of antibodiesare also provided. In some embodiments, a unit dosage is providedwherein the unit dosage contains a predetermined amount of a compositioncomprising an antibody or combination of antibodies, with or without oneor more additional agents. In some embodiments, such a unit dosage issupplied in single-use prefilled syringe for injection. In variousembodiments, the composition contained in the unit dosage may comprisesaline, sucrose, or the like; a buffer, such as phosphate, or the like;and/or be formulated within a stable and effective Ph range.Alternatively, in some embodiments, the composition may be provided as alyophilized powder that may be reconstituted upon addition of anappropriate liquid, for example, sterile water. In some embodiments, thecomposition comprises one or more substances that inhibit proteinaggregation, including, but not limited to, sucrose and arginine. Insome embodiments, a composition of the invention comprises heparinand/or a proteoglycan.

Pharmaceutical compositions are administered in an amount effective fortreatment or prophylaxis of the specific indication. The therapeuticallyeffective amount is typically dependent on the weight of the subjectbeing treated, his or her physical or health condition, theextensiveness of the condition to be treated, or the age of the subjectbeing treated. In general, antibodies may be administered in an amountin the range of about 10 μg/kg body weight to about 100 mg/kg bodyweight per dose. In some embodiments, antibodies may be administered inan amount in the range of about 50 μg/kg body weight to about 5 mg/kgbody weight per dose. In some embodiments, antibodies may beadministered in an amount in the range of about 100 μg/kg body weight toabout 10 mg/kg body weight per dose. In some embodiments, antibodies maybe administered in an amount in the range of about 100 μg/kg body weightto about 20 mg/kg body weight per dose. In some embodiments, antibodiesmay be administered in an amount in the range of about 0.5 mg/kg bodyweight to about 20 mg/kg body weight per dose.

The antibody compositions may be administered as needed to subjects.Determination of the frequency of administration may be made by personsskilled in the art, such as an attending physician based onconsiderations of the condition being treated, age of the subject beingtreated, severity of the condition being treated, general state ofhealth of the subject being treated and the like. In some embodiments,an effective dose of an antibody is administered to a subject one ormore times. In various embodiments, an effective dose of an antibody isadministered to the subject once a month, less than once a month, suchas, for example, every two months or every three months. In otherembodiments, an effective dose of an antibody is administered more thanonce a month, such as, for example, every three weeks, every two weeksor every week. In some embodiments, an effective dose of an antibody isadministered once per 1, 2, 3, 4, or 5 weeks. In some embodiments, aneffective dose of an antibody is administered twice or three times perweek. An effective dose of an antibody is administered to the subject atleast once. In some embodiments, the effective dose of an antibody maybe administered multiple times, including for periods of at least amonth, at least six months, or at least a year.

Additional Combination Therapy

The above therapeutic combinations may be administered alone or withother modes of treatment. They may be provided before, substantiallycontemporaneous with, or after other modes of treatment, for example,surgery, chemotherapy, radiation therapy, or the administration of abiologic, such as another therapeutic antibody. In some embodiments, thecancer has recurred or progressed following a therapy selected fromsurgery, chemotherapy, and radiation therapy, or a combination thereof.

For treatment of cancer, the combinations may be administered inconjunction with one or more additional anti-cancer agents, such as achemotherapeutic agent, growth inhibitory agent, anti-cancer vaccinesuch as a gene therapy vaccine, anti-angiogenesis agent and/oranti-neoplastic composition. Nonlimiting examples of chemotherapeuticagent, growth inhibitory agent, anti-cancer vaccine, anti-angiogenesisagent and anti-neoplastic composition that can be used in combinationwith the antibodies of the present invention are provided herein under“Definitions.”

In some embodiments, an anti-inflammatory drug may be administered withthe combination, such as a steroid or a non-steroidal anti-inflammatorydrug (NSAID).

EXAMPLES

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1: Humanized Anti-CSF1R Antibodies

Various humanized anti-CSF1R antibodies were developed previously. See,e.g., PCT Publication No. WO 2011/140249.

The sequences for each of the humanized heavy chain variable regions andhumanized light chain variable regions, aligned with the sequences ofthe parental chimeric antibody variable regions and the sequences of thehuman acceptor variable framework regions are shown in FIGS. 1 (heavychains) and 2 (light chains). The changes in humanized variable regionsequences relative to the human acceptor variable framework regionsequences are boxed. Each of the CDRs for each of the variable regionsis shown in a boxed region, and labeled as “CDR” above the boxedsequences.

Table 8, below, shows the full sequences for the humanized heavy chainsand humanized light chains of antibodies huAb1 to huAb16. The name andSEQ ID Nos of the humanized heavy chain and humanized light chain ofeach of those antibodies is shown in Table 3.

TABLE 3 Humanized heavy chains and light chains of huAb1 to huAb16Humanized antibody Humanized HC SEQ ID NO Humanized LC SEQ ID NO huAb1h0301-H0 53 h0301-L0 60 huAb2 h0301-H1 54 h0301-L0 60 huAb3 h0301-H2 55h0301-L0 60 huAb4 h0301-H0 53 h0301-L1 61 huAb5 h0301-H1 54 h0301-L1 61huAb6 h0301-H2 55 h0301-L1 61 huAb7 h0302-H1 56 h0302-L0 62 huAb8h0302-H1 56 h0302-L1 63 huAb9 h0302-H1 56 h0302-L2 64 huAb10 h0302-H2 57h0302-L0 62 huAb11 h0302-H2 57 h0302-L1 63 huAb12 h0302-H2 57 h0302-L264 huAb13 h0311-H1 58 h0311-L0 65 huAb14 h0311-H1 58 h0311-L1 66 huAb15h0311-H2 59 h0311-L0 65 huAb16 h0311-H2 59 h0311-L1 66

The 16 humanized antibodies were tested for binding to human, cynomolgusmonkey, and mouse CSF1R ECD, as described previously. See, e.g., PCTPublication No. WO 2011/140249. The antibodies were found to bind toboth human and cynomolgus monkey CSF1R ECD, but not to mouse CSF1R ECD.The humanized antibodies were also found to block binding of CSF1 andIL-34 to both human and cynomolgus CSF1R and to inhibit CSF1-induced andIL-34-induced phosphorylation of human CSF1R expressed in CHO cells.See, e.g., PCT Publication No. WO 2011/140249.

The k_(a), k_(d), and K_(D) for binding to human CSF1R ECD werepreviously determined and are shown in Table 4. See, e.g., PCTPublication No. WO 2011/140249.

!TABLE 4 Humanized antibody binding affinity for human CSF1R huAb k_(a)(M⁻¹s⁻¹) K_(d) (s⁻¹) K_(D) (Nm) huAb 0301-L0H0 3.22 × 10⁶ 1.11 × 10⁻⁰³0.35 huAb 0301-L0H1 3.56 × 10⁶ 1.22 × 10⁻⁰³ 0.34 huAb 0301-L0H2 2.32 ×10⁶ 6.60 × 10⁻⁰⁴ 0.28 huAb 0301-L1H0 3.29 × 10⁶ 1.15 × 10⁻⁰³ 0.35 huAb0301-L1H1 2.87 × 10⁶ 9.21 × 10⁻⁰⁴ 0.32 huAb 0301-L1H2 2.95 × 10⁶ 7.42 ×10⁻⁰⁴ 0.25 huAb 0302-L0H1 3.54 × 10⁶ 3.69 × 10⁻⁰³ 1.04 huAb 0302-L1H13.47 × 10⁶ 4.04 × 10⁻⁰³ 1.17 huAb 0302-L2H1 1.60 × 10⁶ 9.14 × 10⁻⁰⁴ 0.57huAb 0302-L0H2 3.40 × 10⁶ 1.79 × 10⁻⁰³ 0.53 huAb 0302-L1H2 2.71 × 10⁶1.53 × 10⁻⁰³ 0.56 huAb 0302-L2H2 1.84 × 10⁶ 8.40 × 10⁻⁰⁴ 0.46 huAb0311-L0H1 1.22 × 10⁶ 5.40 × 10⁻⁰⁴ 0.44 huAb 0311-L1H1 1.32 × 10⁶ 6.64 ×10⁻⁰⁴ 0.50 huAb 0311-L0H2 1.34 × 10⁶ 4.73 × 10⁻⁰⁴ 0.35 huAb 0311-L1H21.51 × 10⁶ 6.09 × 10⁻⁰⁴ 0.40

Example 2: Enhancement of Anti-Tumor Activity with Combination Therapy

6-8 week old female C57BL/6 mice are housed 5 animals per cage withaccess to food and water ad libitum. Mice are acclimated for at least 3days after arrival in the vivarium. Mice are weighed and their flanksshaved prior to tumor cell line inoculation.

Murine colon adenocarcinoma cell ine MC38 is cultured in RPMI+10% FBS+2mM L-glutamine+antibiotic/antimycotic at 37° C. with 5% CO₂. Cells aresuspended in a solution of 50%/v of DPBS and 50%/v of Matrigel at aconcentration of 5 million cells/ml. 100 μl of cell solution (0.5million cells) are implanted on the right flank of the each mouse usinga 27G1/2 needle. Cells are kept from settling to the bottom of the tubeby using an 18G needle and syringe and by slight vortex. Mice areanesthetized using isofluorane to reduce stress and to allow for moreprecise tumor cell implantation. Length (L) and Width (W) of each tumoris measured using an electronic caliper and the Volume (V) of the tumorwill be calculated using V=(L×W²)/2. Once the mean tumor volume reachesapproximately 105 mm³, mice are grouped and dosed as discussed below.

Groups and Dosing

Mice were separated into the following groups and administered chimericrat anti-mouse CSF1R antibody (mouse IgG1; referred to as “cmFPA008”)and/or anti-CD40 antibody FGK45 (Bio X Cell; see Rolink et al., 1996,Immunity 5:319-330) according to the dosing schedules described below.

-   -   Control: Murine IgG, 30 mg/kg, i.p. 1×/wk, starting on day 0.        Rat IgG 100 μg i.p. day 0.    -   huAb1: anti-CSF1R antibody, 30 mg/kg, i.p. 1×/wk, starting on        day 0. Rat IgG 100 μg i.p. day 0.    -   Anti-CD40 (High): anti-CD40 antibody, 100 μg i.p. on day 0.        Murine IgG, 30 mg/kg, i.p. 1×/wk, starting day 0.    -   Combo (High): anti-CSF1R antibody, 30 mg/kg, i.p. 1×/wk,        starting on day 0, Anti-CD40, 100 μg i.p. day 0.    -   Anti-CD40 (Low): anti-CD40 antibody, 30 μg i.p. on day 0. Murine        IgG, 30 mg/kg, i.p. 1×/wk, starting day 0.    -   Combo (Low): anti-CSF1R antibody, 30 mg/kg, i.p. 1×/wk, starting        on day 0, anti-CD40 antibody, 30 μg i.p. day 0.

The dosing schedule and groupings are shown in Table 5:

TABLE 5 Study dosing and grouping Dosing Dosing Treatment #1: (mg/kg,Treatment #2: (mg/kg, Mice Group chimeric Ab1 schedule) Anti-CD40schedule) (n) 1 Murine IgG 30, 1× Rat IgG 100 μg on Day 0 10 2anti-CSF1R week (qw) 10 antibody 3 Murine IgG anti-CD40 100 μg on Day 010 4 anti-CSF1R antibody 10 antibody 5 Murine IgG  30 μg on Day 0 10 6anti-CSF1R 10 antibody

Animals are euthanized prior to the end of the study if any of thefollowing signs are observed:

-   -   Body weight loss of equal or greater than 15% of initial body        weight.    -   Tumor ulceration is observed.    -   Mice appear moribund.    -   Individual tumor volume is equal or larger than 10% of initial        body weight

Plasma is collected for pharmacokinetic (PK) analysis. On the final dayof the study, whole blood is collected via intra-cardiac bleeds, andplasma is isolated for PK analysis (bioanalytical group). At least five(5) tumors from each group are collected for the following analyses.Single-cell isolates of the tumors are generated by collagenasetreatment, and FACS is used to examine the infiltration of immune cellsinto the tumor(s). Tumor sections are also snap frozen in liquidnitrogen and stored at −80° C. for protein and mRNA extraction. Tumorsections are embedded in Optimum Cutting Temperature compound (OCT) andstored in −80° C. Tumor sections are also placed in 10% bufferedformalin overnight, and then transferred to 70% ethanol the followingday.

The results of this experiment are shown in FIGS. 3 and 4 . As shown inFIG. 3 , the combination of anti-CSF1R antibody and anti-CD40 antibody(high) resulted in early tumor regression followed by tumor stasis. Thecombination of anti-CSF1R antibody and anti-CD40 antibody (low) alsodemonstrated greater efficacy than either treatment alone. FIG. 4 showsindividual tumor volumes at (A) day 11 and (B) day 13. Table 6 shows thetumor volume statistics at day 11 and day 13 using one-way ANOVA. Thecombination of anti-CSF1R antibody and anti-CD40 antibody (high) showedsignificantly better efficacy than either treatment alone.

TABLE 6 Tumor volume statistics Day 11 Day 13 Mean Mean Group TV ± SD %TGI p-value TV ± SD % TGI p-value IgG/IgG 729.3 ± 183.8 0.00 — 942.4 ±278.3 0.00 — anti-CSF1R 579.2 ± 87.87 20.58  0.0037 710.0 ± 80.05 24.660.0004 antibody/IgG vs Combo (Low) IgG/anti-CD40 452.5 ± 111.9 37.950.5888 ns 645.2 ± 162.0 31.53 0.0139 antibody (low) vs Combo (Low)IgG/anti-CD40 450.8 ± 105.6 38.18 <0.0001 650.6 ± 151.5 30.96 <0.0001antibody (High) vs Combo (High) Combo (High) 157.4 ± 55.00 78.41 <0.0001223.3 ± 98.32 76.31 <0.0001 vs anti-CSF1R antibody/IgG

FIG. 5 shows body weight for all animals in the study, measured at leasttwice per week. No significant different in weight was observed for anygroup relative to control.

Table of Sequences

Table 10 provides certain sequences discussed herein. All polypeptideand antibody sequences are shown without leader sequences, unlessotherwise indicated.

TABLE 10 Sequences and Descriptions SEQ ID NO Description Sequence  1hCSF1R IPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL(full-length, STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFEno leader DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKFsequence) IQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEAAQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVDFQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGLNLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLPRLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLCAASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQSLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHTHP PDEFLFTPVVVACMSIMALL LLLLLLLLYK YKQKPKYQVR WKIIESYEGN SYTFIDPTQLPYNEKWEFPR NNLQFGKTLG AGAFGKVVEA TAFGLGKEDA VLKVAVKMLKSTAHADEKEA LMSELKIMSH LGQHENIVNL LGACTHGGPV LVITEYCCYGDLLNFLRRKA EAMLGPSLSP GQDPEGGVDY KNIHLEKKYV RRDSGFSSQGVDTYVEMRPV STSSNDSFSE QDLDKEDGRP LELRDLLHFS SQVAQGMAFLASKNCIHRDV AARNVLLTNG HVAKIGDFGL ARDIMNDSNY IVKGNARLPVKWMAPESIFD CVYTVQSDVW SYGILLWEIF SLGLNPYPGI LVNSKFYKLVKDGYQMAQPA FAPKNIYSIM QACWALEPTH RPTFQQICSF LQEQAQEDRRERDYTNLPSS SRSGGSGSSS SELEEESSSE HLTCCEQGDI AQPLLQPNNY QFC  2 hCSF1RMGPGVLLLLL VATAWHGQGI PVIEPSVPEL VVKPGATVTL RCVGNGSVEW (full-length, + DGPPSPHWTL YSDGSSSILS TNNATFQNTG TYRCTEPGDP LGGSAAIHLY leaderVKDPARPWNV LAQEVVVFED QDALLPCLLT DPVLEAGVSL VRVRGRPLMR sequence)HTNYSFSPWH GFTIHRAKFI QSQDYQCSAL MGGRKVMSIS IRLKVQKVIPGPPALTLVPA ELVRIRGEAA QIVCSASSVD VNFDVFLQHN NTKLAIPQQSDFHNNRYQKV LTLNLDQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAYLNLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPKLANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRYPPEVSVIWTF INGSGTLLCA ASGYPQPNVT WLQCSGHTDR CDEAQVLQVWDDPYPEVLSQ EPFHKVTVQS LLTVETLEHN QTYECRAHNS VGSGSWAFIPISAGAHTHPP DEFLFTPVVV ACMSIMALLL LLLLLLLYKY KQKPKYQVRWKIIESYEGNS YTFIDPTQLP YNEKWEFPRN NLQFGKTLGA GAFGKVVEATAFGLGKEDAV LKVAVKMLKS TAHADEKEAL MSELKIMSHL GQHENIVNLLGACTHGGPVL VITEYCCYGD LLNFLRRKAE AMLGPSLSPG QDPEGGVDYKNIHLEKKYVR RDSGFSSQGV DTYVEMRPVS TSSNDSFSEQ DLDKEDGRPLELRDLLHFSS QVAQGMAFLA SKNCIHRDVA ARNVLLTNGH VAKIGDFGLARDIMNDSNYI VKGNARLPVK WMAPESIFDC VYTVQSDVWS YGILLWEIFSLGLNPYPGIL VNSKFYKLVK DGYQMAQPAF APKNIYSIMQ ACWALEPTHRPTFQQICSFL QEQAQEDRRE RDYTNLPSSS RSGGSGSSSS ELEEESSSEHLTCCEQGDIA QPLLQPNNYQ FC  5 hCSF1RIPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL ECD.506STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFEDQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKFIQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEAAQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVDFQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGLNLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLPRLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLCAASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQSLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAH  6 hCSF1RIPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL ECD.506-FcSTNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFEDQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKFIQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEAAQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVDFQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGLNLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLPRLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLCAASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQSLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHEPK SSDKTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK  7 cynoCSF1RMGPGVLLLLL VVTAWHGQGI PVIEPSGPEL VVKPGETVTL RCVGNGSVEW ECD (withDGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY leaderVKDPARPWNV LAKEVVVFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR sequence)HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIPGPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRSDFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAYLDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPKLANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRYPPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR CDEAQVLQVWVDPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIP ISAGAR  8cynoCSF1R MGPGVLLLLL VVTAWHGQGI PVIEPSGPEL VVKPGETVTL RCVGNGSVEW ECD-FcDGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY (with leaderVKDPARPWNV LAKEVVVFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR sequence)HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIPGPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRSDFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAYLDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPKLANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRYPPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR CDEAQVLQVWVDPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIPISAGARGSEP KSSDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK  3 Light chainMETDTLLLWV LLLWVPGSTG leader  sequence  4 Heavy chainMAVLGLLLCL VTFPSCVLS leader sequence  9 Fab 0301 heavyEVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD chain variable INPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARES regionPYFSNLYVMD YWGQGTSVTV SS 10 Fab 0301 lightNIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL chain variableLIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLS regionTFGGGTKLEI K 11 Fab 0302 heavyEIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY chain variableINPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYF regionDGTFDYALDY WGQGTSITVS s 12 Fab 0302 lightDVVVTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL chain variableLIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPW regionTFGGGTRLEI K 13 Fab 0311 heavyEIQLQQSGPD LMKPGASVKM SCKASGYIFT DYNMHWVKQN QGKSLEWMGE chain variableINPNNGVVVY NQKFKGTTTL TVDKSSSTAY MDLHSLTSED SAVYYCTRAL regionYHSNFGWYFD SWGKGTTLTV SS 14 Fab 0311 lightDIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL chain variableLIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPW regionTFGGGTRLEI K 15 0301 heavy GYTFTDNYMI chain CDR1 16 0301 heavyDINPYNGGTT FNQKFKG chain CDR2 17 0301 heavy ESPYFSNLYV MDY chain CDR3 180301 light KASQSVDYDG DNYMN chain CDR1 19 0301 light AASNLEs chain CDR220 0301 light HLSNEDLST chain CDR3 21 0302 heavy GYTFSDFNIH chain CDR122 0302 heavy YINPYTDVTV YNEKFKG chain CDR2 23 0302 heavy YFDGTFDYAL DYchain CDR3 24 0302 light RASESVDNYG LSFMN chain CDR1 25 0302 lightTASNLES chain CDR2 26 0302 light QQSKELPWT chain CDR3 27 0311 heavyGYIFTDYNMH chain CDR1 28 0311 heavy EINPNNGVVV YNQKFKG chain CDR2 290311 heavy ALYHSNFGWY FDS chain CDR3 30 0311 light KASQSVDYDG DSHMNchain CDR1 31 0311 light TASNLES chain CDR2 32 0311 light QQGNEDPWTchain CDR3 33 cAb 0301EVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD heavy chainINPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARESPYFSNLYVMD YWGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 34 cAb 0301NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL light chainLIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLSTFGGGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 35 cAb 0302EIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYFDGTFDYALDY WGQGTSITVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 36 cAb 0302DVVVTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL light chainLIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPWTFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 37 cAb 0311EIQLQQSGPD LMKPGASVKM SCKASGYIFT DYNMHWVKQN QGKSLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSSSTAY MDLHSLTSED SAVYYCTRALYHSNFGWYFD SWGKGTTLTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 38 cAb 0311DIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL light chainLIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPWTFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 39 h0301-H0QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARES variablePYFSNLYVMD YWGQGTLVTV SS region 40 h0301-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARES variablePYFSNLYVMD YWGQGTLVTV SS region 41 h0301-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD heavy chainINPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARES variablePYFSNLYVMD YWGQGTLVTV SS region 42 H0302-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWMGY heavy chainINPYTDVTVY NEKFKGRVTI TSDKSTSTAY MELSSLRSED TAVYYCASYF variableDGTFDYALDY WGQGTLVTVS S region 43 H0302-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYF variableDGTFDYALDY WGQGTLVTVS S region 44 H0311-H1QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRAL variableYHSNFGWYFD SWGQGTLVTV SS region 45 H0311-H2QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRAL variableYHSNFGWYFD SWGQGTLVTV SS region 46 h0301-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS variableTFGGGTKVEI K region 47 h0301-L1NIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS variableTFGGGTKVEI K region 48 H0302-L0EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQSKELPW variableTFGQGTKVEI K region 49 H0302-L1EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW variableTFGQGTKVEI K region 50 H0302-L2EIVVTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW variableTFGQGTKVEI K region 51 H0311-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPW variableTFGQGTKVEI K region 52 H0311-L1DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPW variableTFGQGTKVEI K region 53 h0301-H0QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARESPYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 54 h0301-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARESPYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 55 h0301-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD heavy chainINPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARESPYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 56 H0302-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWMGY heavy chainINPYTDVTVY NEKFKGRVTI TSDKSTSTAY MELSSLRSED TAVYYCASYFDGTFDYALDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 57 H0302-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYFDGTFDYALDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 58 H0311-H1QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRALYHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 59 H0311-H2QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRALYHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 60 h0301-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLSTFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 61 h0301-L1NIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLSTFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 62 H0302-L0EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQSKELPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 63 H0302-L1EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 64 H0302-L2EIVVTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 65 H0311-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 66 H0311-L1DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 67 Human CSF1EEVSEYCSHM IGSGHLQSLQ RLIDSQMETS CQITFEFVDQ EQLKDPVCYLKKAFLLVQDI MEDTMRFRDN TPNAIAIVQL QELSLRLKSC FTKDYEEHDKACVRTFYETP LQLLEKVKNV FNETKNLLDK DWNIFSKNCN NSFAECSSQG HERQSEGS 68Human IL-34 NEPLEMWPLT QNEECTVTGF LRDKLQYRSR LQYMKHYFPI NYKISVPYEGVFRIANVTRL QRAQVSEREL RYLWVLVSLSATESVQDVLL EGHPSWKYLQEVQTLLLNVQ QGLTDVEVSP KVESVLSLLN APGPNLKLVR PKALLDNCFRVMELLYCSCC KQSSVLNWQD CEVPSPQSCS PEPSLQYAAT QLYPPPPWSPSSPPHSTGSV RPVRAQGEGL LP 69 Human QVQLVQSGAE VKKPGSSVKV SCKAS acceptor AFR1 70 Human WVRQAPGQGL EWMG acceptor A  FR2 71 HumanRVTITADKST STAYMELSSL RSEDTAVYYC AR acceptor A FR3 72 Human WGQGTLVTVS Sacceptor A FR4 73 Human QVQLVQSGAE VKKPGSSVKV SCKAS acceptor B FR1 74Human WVRQAPGQGL EWMG acceptor B FR2 75 HumanRVTITADKST STAYMELSSL RSEDTAVYYC AR acceptor B FR3 76 Human WGQGTLVTVSSacceptor B FR4 77 Human QVQLVQSGAE VKKPGSSVKV SCKAS acceptor C FR1 78Human WVRQAPGQGL EWMG acceptor C FR2 79 HumanRVTITADKST STAYMELSSL RSEDTAVYYC AR acceptor C FR3 80 Human WGQGTLVTVS Sacceptor C FR4 81 Human EIVLTQSPAT LSLSPGERAT LSC acceptor D FR1 82Human WYQQKPGQAP RLLIY acceptor D FR2 83 HumanGIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC acceptor D FR3 84 Human FGGGTKVEIKacceptor D FR4 85 Human EIVLTQSPAT LSLSPGERAT LSC acceptor E FR1 86Human WYQQKPGQAP RLLIY acceptor E FR2 87 HumanGIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC acceptor E FR3 88 Human FGQGTKVEIKacceptor E FR4 89 Human EIVLTQSPAT LSLSPGERAT LSC acceptor F FR1 90Human WYQQKPGQAP RLLIY acceptor F FR2 91 HumanGIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC acceptor F FR3 92 Human FGQGTKVEIKacceptor F FR4 93 mCSF1RAPVIEPSGPE LVVEPGETVT LRCVSNGSVE WDGPISPYWT LDPESPGSTL ECD-FcTTRNATFKNT GTYRCTELED PMAGSTTIHL YVKDPAHSWN LLAQEVTVVEGQEAVLPCLI TDPALKDSVS LMREGGRQVL RKTVYFFSPW RGFIIRKAKVLDSNTYVCKT MVNGRESTST GIWLKVNRVH PEPPQIKLEP SKLVRIRGEAAQIVCSATNA EVGFNVILKR GDTKLEIPLN SDFQDNYYKK VRALSLNAVDFQDAGIYSCV ASNDVGTRTA TMNFQVVESA YLNLTSEQSL LQEVSVGDSLILTVHADAYP SIQHYNWTYL GPFFEDQRKL EFITQRAIYR YTFKLFLNRVKASEAGQYFL MAQNKAGWNN LTFELTLRYP PEVSVTWMPV NGSDVLFCDVSGYPQPSVTW MECRGHTDRC DEAQALQVWN DTHPEVLSQK PFDKVIIQSQLPIGTLKHNM TYFCKTHNSV GNSSQYFRAV SLGQSKQEPK SSDKTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK 94 Human IgG4ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV S241PHTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVESKYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGK 95 Human IgκRTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSGNSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 96human CD40 MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSL CQPGQKLVSDprecursor CTEFTETECL PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD(with signal TICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD TICEPCPVGFsequence) FSNVSSAFEK CHPWTSCETK DLVVQQAGTN KTDVVCGPQD RLRALVVIPIUniProtKB/ IFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPSwiss-Prot: VQETLHGCQP VTQEDGKESR ISVQERQ P25942.1, 4 Mar. 2015 97human CD40 EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECL PCGESEFLDT(mature, WNRETHCHQH KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCVwithout signal LHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEK CHPWTSCETKsequence) DLVVQQAGTN KTDVVCGPQD RLRALVVIPI IFGILFAILL VLVFIKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 98Dacetuzumab EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVARheavy chain VIPNAGGTSY NQKFKGRFTL SVDNSKNTAY LQMNSLRAED TAVYYCAREGIYWWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEPVTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVNHKPSNTKVDK KVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMISRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPPSREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK 99 DcetuzumabDIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK light chainLLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YFCSQTTHVPWTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAKVQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACEVTHQGLSSPV TKSFNRGEC

The invention claimed is:
 1. A method of treating cancer in a subject comprising administering to the subject an anti-CSF1R antibody and an agonist anti-CD40 antibody, wherein the anti-CSF1R antibody binds human CSF1R and is selected from: a) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 39 and a light chain comprising the sequence of SEQ ID NO: 46; b) an antibody comprising a heavy chain comprising a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15, an HC CDR2 having the sequence of SEQ ID NO: 16, and an HC CDR3 having the sequence of SEQ ID NO: 17, and a light chain comprising a light chain (LC) CDR1 having the sequence of SEQ ID NO: 18, a LC CDR2 having the sequence of SEQ ID NO: 19, and a LC CDR3 having the sequence of SEQ ID NO: 20; and c) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 53 and a light chain comprising the sequence of SEQ ID NO:
 60. 2. The method of claim 1, wherein the anti-CD40 antibody comprises the CDRs of an antibody selected from CP-870,893; dacetuzumab; SEA-CD40; ADC-1013; R07009789; and Chi Lob 7/4.
 3. The method of claim 2, wherein the anti-CD40 antibody comprises the heavy chain and light chain variable regions of an antibody selected from CP-870,893; dacetuzumab; SEA-CD40; ADC-1013; R07009789; and Chi Lob 7/4.
 4. The method of claim 3, wherein the anti-CD40 antibody is an antibody selected from CP-870,893; dacetuzumab; SEA-CD40; ADC-1013; R07009789; and Chi Lob 7/4.
 5. The method of claim 1, wherein the anti-CSF1R antibody and the anti-CD40 antibody are administered concurrently or sequentially.
 6. The method of claim 1, wherein the anti-CSF1R antibody and the anti-CD40 antibody are administered concurrently.
 7. The method of claim 5, wherein one or more doses of the anti-CD40 antibody are administered prior to administering an anti-CSF1R antibody.
 8. The method of claim 5, wherein one or more doses of the anti-CSF1R antibody are administered prior to administering the anti-CD40 antibody.
 9. The method of claim 1, wherein the cancer is selected from non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, endometrial cancer, Hodgkin's lymphoma, lung cancer, glioma, glioblastoma multiforme, colon cancer, breast cancer, bone cancer, skin cancer, uterine cancer, gastric cancer, stomach cancer, lymphoma, lymphocytic leukemia, multiple myeloma, prostate cancer, mesothelioma, and kidney cancer.
 10. The method of claim 1, wherein the cancer is recurrent or progressive after a therapy selected from surgery, chemotherapy, radiation therapy, or a combination thereof.
 11. The method of claim 1, wherein the anti-CSF1R antibody blocks binding of CSF1 and/or IL-34 to CSF1R.
 12. The method of claim 1, wherein the anti-CSF1R antibody inhibits ligand-induced CSF1R phosphorylation in vitro.
 13. The method of claim 1, wherein the antibody is a humanized antibody.
 14. The method of claim 1, wherein the antibody is selected from a Fab, an Fv, an scFv, a Fab′, and a (Fab′)₂.
 15. The method of claim 1, wherein the anti-CSF1R antibody comprises a human IgG4 heavy chain constant region with an S241P mutation.
 16. The method of claim 1, wherein the anti-CSF1R antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 39 and a light chain comprising the sequence of SEQ ID NO:
 46. 17. The method of claim 16, wherein the anti-CSF1R antibody comprises a human IgG4 heavy chain constant region with an S241P mutation.
 18. The method of claim 1, wherein the anti-CSF1R antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 53 and a light chain comprising the sequence of SEQ ID NO:
 60. 